Poster Presentation
SOT 64th Annual Meeting and ToxExpo
Orlando - March 16-20 2025
Abstract:
Due to well publicised human health and environmental concerns, the State of Washington Department of Ecology has defined criteria for safer alternatives to N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) and its ozonation transformation products (State of Washington Department of Ecology, 2023). 6PPD is an antioxidant and antiozonant that prevents rubber from degrading and reacting with oxygen (O2) and ozone (O3). It is widely used in the motor vehicle tire and general rubber goods sector. Washington State Department of Ecology considers 6PPD as a toxicant of high concern due to aquatic toxicity, reproductive toxicity, environmental persistence, and bioaccumulation potential.
Here we present the results of human health hazard testing of N1,N4-dicyclohexylbenzene-1,4-diamine (CCPD, CAS No. 4175-38-6), up to and including reproductive and developmental toxicity screening in accordance with OECD TG 422 (the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test). CCPD is an innovation substance designed as a replacement for 6PPD using safe by design concepts. The testing strategy considered both requirements for registration under relevant regulatory jurisdictions (e.g., EU REACH), and the 6PPD Alternatives Assessment Hazard criteria.
The acute oral toxicity of CCPD was confirmed via an OECD TG 423 as Acute Tox. 4, with an LD50 of 500 mg/kg bw. An acute dermal toxicity study according to OECD TG 402 confirmed no GHS classification, with an LD50 >2,000 mg/kg d. This classification is in line with that of 6PPD, which is also classified at Acute Tox. 4. Unlike 6PPD for which clastogenicity was reported in vitro, CCPD was found to have no mutagenic, clastogenic or aneugenic potential as demonstrated in OECD TG 471, OECD TG 487 and OECD TG 490 assays. In a profile similar to that of 6PPD, CCPD is Eye irritant 2 and skin sensitiser 1A according to the results of OECD TG 492B and OECD TG 442C assays and accompanying GHS criteria.
In the OECD TG 422 study, CCPD was administered orally via gavage to Wistar Hans rat (Charles River Deutschland, Sulzfeld, Germany) to evaluate systemic endpoints, male and female reproductive performance and development of the offspring. F0 males were dosed for a minimum of 28 days, including 2 weeks prior to mating. F0 females were dosed for 2 weeks prior to mating, through gestation and up to at least lactation Day 13.
A 10-day dose range finding study established the high dose for the main study at 115 mg/kg/day, based on dose limiting toxicity at ≥150 mg/kg/day, (mortality, significant reduction in bodyweight and food consumption, clinical observations) and moderate toxicity without dose limiting features at 75 mg/kg/day. Dose levels of 60 mg/kg bw/day and 20 mg/kg bw/day were selected as mid- and low-dose level, respectively. These observations are not in concordance with those for 6PPD, where different clinical signs were observed and far lower doses were required due to toxicity observed.
In the definitive study, the results indicated some generalised toxicity at the higher dose levels, manifested in F0 males by a reduction in bodyweight with concomitant reduction in food consumption. In F0 females, an initial bodyweight loss which correlated with reduction in food consumption was followed by recovery during gestation and lactation (although bodyweight gain remained reduced vs. control). In contrast, for 6PPD mean body weights, body weight gains and food consumption throughout the treatment period (including during gestation and lactation for females) were unaffected by test substance administration at all dosage levels (in both the OECD 443 and importantly, OECD 421, which tested up to a high dose at a similar level to that used in this study), demonstrating further differences between the two chemicals for repeated dose toxicity.
Haematology and clinical chemistry revealed minimal variation vs. control. There was a slight reduction in mean corpuscular volume and haemoglobin values in both sexes. Similar changes (across these and other values) were seen with 6PPD, all being attributed to natural variation within the species rather than test item.
In male and female animals there was a dose-dependent decrease in absolute and relative liver weight, which presented without histological correlate. In 6PPD, liver findings in several repeated dose toxicity studies (sub-acute to chronic) included increased weight, hepatocellular vacuolation and periportal fatty changes were considered overall to be adverse and used as the basis for selection of dose descriptor for derivation of DNELs. As such, the adverse liver effects identified for 6PPD are not shared with CCPD. In female animals, a reduction in absolute and relative ovary weight was observed at the highest dose tested, without histological correlate or functional impact on reproduction.
In relation to reproductive parameters, at 115 mg/kg bw/day mating, precoital, fertility and pregnancy indices were comparable to control. There was a slightly lower number of implantations when compared to the control. However, based on review of the individual data this was considered to be within the range of normal variation and not related to the test item. As such, there was considered to be no effect on reproduction based on the parameters evaluated. Given that the primary effect of 6PPD was dystocia and prolonged labour, with multiple total litter losses as a result, these findings are extremely positive for CCPD as a potentially safer alternative.
In the highest dose tested only, pup bodyweight was lower across all measurements to postnatal day 13. This is in accordance with but less severe than 6PPD, where bodyweight reductions were more pronounced at lower doses (and in more of the dose groups). Physical development evaluations revealed no alteration to AGD, nor alteration in thyroid hormone levels at postnatal day 14-16. Overall, there was minimal effect on development based on the parameters evaluated. Reproductive and developmental parameters will be evaluated in further detail within future planned testing.
The results to date indicate that CCPD may present a favourable alternative to 6PPD. The studies conducted meet internationally recognised requirements for endpoint coverage under relevant regulatory jurisdictions for CCPD’s registration, and the aforementioned critical Alternatives Assessment Hazard Criteria. Future work will include an OECD 414 prenatal developmental toxicity test, and an OECD 443 extended one generation reproductive toxicity study (EOGRTS) to conclusively evaluate the reproductive and developmental toxicity of CCPD.
Presenter Bio: Dr. Bryony Ross
Bryony is a principal toxicologist & team leader for Human Safety within the Blue Frog Scientific Group of Companies. An accomplished toxicologist with over 15 years’ experience and a successful track record, her areas of expertise are chemical hazard assessment, reproductive and developmental toxicology, endocrine disruption and nanotoxicology. Bryony is passionate about developing intelligent approaches for the testing and assessment of hazards, sound scientific communication & animal welfare / the 3Rs principles.
