Reaction mass of 3-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-hydroxy-4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In the one-generation study with rats, the NOAEL for parental toxicity was set at 25 mg/kg bw/day, based on gestation length increase in the 100 and 500 mg/kg bw dose groups. There was no adverse effect on fertility, therefore the NOAEL for fertility is set at the highest dose level, 500 mg/kg bw/day. The NOAEL for developmental effects was set at 25 mg/kg bw/day, based on the skin effects (skin peeling and flaking, and acanthosis and hyperkeratosis observed at necropsy) in male and female F1 animals at 100 mg/kg bw.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The selected study is a guideline study in compliance with GLP and has Klimisch score of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity of the test substance was studied in a study with Sprague-Dawley rats, conducted according to OECD Guideline 415 in compliance with GLP. The substance was administered at dose levels of 0, 25, 100 and 500 mg/kg bw/day as a solution in arachis oil to groups of 24 rats/sex/dose for 76 days pre-mating and during maximum 21 days mating, after which males were killed, while females and subsequent offspring were killed and examined on day 21 post-partum. Non-pregnant females were killed and examined after day 25 post-coitum. One male treated with 500 mg/kg bw/day was killed in extremis on day 93. One female from this treatment group was found dead on day 97 and a further two females were killed in extremis on days 99 and 100 following difficulties during parturition. Episodes of hunched posture, pilo-erection and tiptoe gait were evident in 500 mg/kg bw/day females during the final week of gestation. Body weight gain for males of the 500 mg/kg bw/day group was generally lower (average -22 %, range -9 % to -43 %) than control animals throughout much of the treatment period, with statistical differences observed in weeks 4, 5, 6 and 10. Females treated with 500 mg/kg bw/day showed a notable reduction in food consumption (average -21 %, range -17 % to -28 %) throughout lactation, with statistically significant differences throughout 3 weeks. There were no treatment-related effects on female estrous cycles or on the type or proportion of females with an anomalous estrous cycle, or toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts. Mating performance was good in all groups with the majority of animals mating within the first four days of pairing. Subsequent pregnancy rate was unaffected by treatment with only 1, 1, 1 and 2 females failing to achieve pregnancy in the control, 25, 100 and 500 mg/kg bw/day groups, respectively. An increased gestation length was observed in the 100 and 500 mg/kg bw dose groups. At necropsy, the adult male treated with 500 mg/kg bw/day that was killed in extremis showed gaseous distension in the gasto-intestinal tract. The female treated with 500 mg/kg bw/day that was found dead around parturition had 21 foetuses in-utero. The two females from this treatment group that were killed in extremis both had dead/inactive fetuses in-utero and red/brown staining around the ano-genital region and dark contents in the stomach or enlarged adrenals and an absent rougae on the non-glandular region of the stomach. No treatment-related microscopic changes were observed in the parental animals. Based on the observed increased gestation length, the NOAEL for parental toxicity was set at 25 mg/kg bw/day.

Life birth index for 500 mg/kg bw/day females was significantly lower (-15 %) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1. Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters. A delay in the onset (+ 2.1 days) and completion (+ 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning. Body weight gain in offspring at 500 mg/kg bw/day was lower (-58 %) than in control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation. Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage. Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day. Based on the skin effects in F1 offspring, the NOAEL was set at 25 mg/kg bw/day for the F1 generation. There was no adverse effect on fertility, therefore the NOAEL for fertility is set at the highest dose level, 500 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

The key study for developmental toxicity is the one-generation study. In this study a NOAEL of 25 mg/kg bw is derived. At higher doses in this study effect on delayed development of the pups were seen and on effects on skin, such as skin peeling were observed. Because of these latter effects are rare two additional studies were performed, which did not lead to a change in NOAEL.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The selected study is a study in compliance with GLP and has Klimisch score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

One-generation study, developmental effects

See for methods and results on repeated dose systemic toxicity the section on fertility above.

Life birth index for 500 mg/kg bw/day females was significantly lower (-15%) than control animals with litter size continuing to be statistically lower than control animals throughout lactation. Of the high dose females that gave birth to live litters, six females had a total litter loss, predominantly between birth and day 1. Skin sloughing was detected in offspring during the first week of lactation in all treatment groups (more pronounced in the high dose group) together with multiple ridges along the tail in 500 and 100 mg/kg bw/day litters. Swollen ears became apparent in 500 and 100 mg/kg bw/day litters together with the premature opening of eyes and sparse fur coverage in 500 mg/kg bw/day litters. A delay in the onset (+ 2.1 days) and completion (+ 2.8 days) of pinna unfolding was evident in 500 mg/kg bw/day offspring together with a reduction in the number of offspring passing surface righting, air righting and pupil reflex. A total of eight 500 mg/kg bw/day litters had not fully completed eye opening by weaning. Body weight gain in offspring at 500 mg/kg bw/day was lower (-58%) than in control animals for the first week of age and again from day 14 to weaning (day 21 of age) (-17%). Litter weight, at this treatment group, was notably lower than control animals throughout lactation. Offspring from females treated with 500 and 100 mg/kg bw/day showed skin sloughing at necropsy. Offspring from females treated with 500 mg/kg bw/day also showed sparse fur coverage. Acanthosis and hyperkeratosis were seen in relation to treatment for the skin of male and female F1 generation animals treated with 500 and 100 mg/kg bw/day. Based on the skin effects in F1 offspring, the NOAEL was set at 25 mg/kg bw/day for the F1 generation.

 

A new study was designed to determine the potential for adverse effects on the offspring observed in the one-generation reproductive study. It was designed to evaluate:

1) whether the pup dermal effects observed in the one-generation reproductive toxicity study were due to pre-or post-natal exposure to the test substance;

2) whether the test substance produced a functional zinc deficiency in the dams, thereby producing skin sloughing, peeling and/or flaking in pups. Fifty female rats were randomly assigned to 6 dosage groups:

Dose groups	Doses in mg/kg bw	Number of rats	Dose administration
Ia	0 (vehicle)	10	GD 0 through 21 or 24
Ib	0 (vehicle)	10	LD 1 through 21
II	500	10	GD through 21 or 24
III	500	10	LD 1 through 21
IV	0 (vehicle)	5	GD 0 through 14
V	500	5	GD 0 through 14

Results: Administration of the test substance at 500 mg/kg bw/day caused mortality as a result of dystocia and adverse clinical signs in the dams treated during the gestation period (Group II). Reductions in body weight gain occurred during the gestation treatment period; although not statistically significant, the reductions were substantial enough (90.3% of the concurrent vehicle control group value) to be toxicologically important. No apparent effects of the test substance on body weight gain were noted during the lactation period, following the cessation of test article administration. At sacrifice, the average terminal body weight was significantly increased, reflecting significant increases in maternal body weight gains after treatment was stopped. Serum chemistry parameters during early lactation, specifically glucose levels, blood urea nitrogen levels and alkaline phosphatase levels and the albumin to globulin ratio were significantly increased by 500 mg/kg bw/day test substance(Group II). Treatment of the dams during the lactation period (Group III) resulted in increased liver weights, body weights (with fluctuating periods of body weight gain and body weight loss) and terminal weights. Cholesterol levels were lower in dams treated during the lactation period (Group III), while creatinine, aspartate aminotransferase, inorganic phosphate levels and albumin to globulin ratios were significantly increased in these dams. Treatment of dams with the test substance during gestation or lactation had no biologically important effect on zinc or metallothionein levels. Pups growth and viability was affected by the treatment during the gestation period (Group II), in that increased incidences of stillbirths occurred as well as pup deaths near parturition and up to lactation day 3. As a result, the viability index was significantly reduced, the lactation index was reduced, and live litter sizes were significantly lower than control group values (Group Ia). Transient reductions in pup weights were noted in the F1 generation as a result of maternal treatment with the test substance during the gestation period (Group II). Clinical signs of cold to touch, often associated with pup mortality, and transient observations of flaking occurred in F1 generation pups. These effects were observed at a dose that caused reduced maternal body weight gains and maternal mortality. Observations in the F1 generation from dams treated during lactation (Group III) included significantly reduced viability and lactation indices, reductions in pup weights that were sustained until sacrifice, compared to the vehicle control (Group Ib), and persistent observations of skin peeling in all litters. In conclusion, when pregnant rats were treated with the test substance during the gestation period, transient clinical signs of flaking were noted in the F1 generation pups with relatively few observations of skin peeling. This effect occurred at maternally toxic levels. Conversely, treatment of the dams with the test substance during the lactation period resulted in skin peeling in all F1 generation pups, without resolution, and with minimal observations of flaking prior to sacrifice.

Study 2: In order to determine if the effects observed in the offspring in abovementioned study were related to the test material transmission through maternal milk and if so, whether the effect was reversible following weaning, another study in compliance with GLP was conducted (Charles River Laboratories, 2009b).

Method: Groups of 10 pregnant female rats were allowed to deliver the litters and then administered the test substance at dose levels of 0, 10, 25 and 500 mg/kg bw/day on days 1-15 or 1-22 post-partum. The concentrations of the test substance in breast milk were evaluated on post-partum days 14 and 21. The pups were allowed to wean and observed for 64 to 67 days post-partum to determine whether skin effects observed in two earlier studies were reversible.

Results: At 500 mg/kg bw/day caused transient reductions in maternal body weight gain early on during the lactation treatment period. At 500 mg/kg bw/day, maternal body weight gains at the end of the lactation treatment period (days post-partum (DPs) 14 to 22) were increased relative to the vehicle control group value, such that the overall body weight gains for the entire lactation period were 25% greater than the vehicle controls. The test substance also reduced maternal absolute and relative feed consumption values in the 500 mg/kg bw/day dosage group for the entire lactation period, as compared to vehicle controls. The test substance was quantifiable in the milk of two of 10 rats on DP 14 and four of five rats on DP 21 in the 500 mg/kg bw/day dosage group. No test substance was detected in the milk of rats treated with 0 (Vehicle), 10 or 25 mg/kg bw/day on either DP 14 or 21. The observed effects in maternal animals are considered to be adverse, the dose level of 500 mg/kg bw/day is considered to be a LOAEL for maternal toxicity. Results F1 generation: Observations in the F1 generation from dams treated with 500 mg/kg bw/day test substance during the lactation period included mortality, reductions in pup body weights and observations of skin peeling. The reductions in pup body weights continued into the post-weaning period for both F1 male and female rats, but did not affect the terminal body weight or the cumulative body weight gains during the post-weaning period. These reductions during the post-weaning period reflected the effects of the test substance on pup body weights that occurred prior to weaning. The observation of skin peeling occurred in all litters in the 500 mg/kg bw/day maternal dosage group as early as DP 8 and, in general, persisted into the post-weaning recovery period until recovery by DP 35. Skin peeling during the post-weaning period occurred more frequently in the F1 female rats than in the F1 male rats. All 36 F1 female rats in the 500 mg/kg bw/day dosage group had the observation on one or more occasions during the post-weaning period, while only 11 of 22 male rats had the observation during this same period. Skin peeling in the 500 mg/kg bw/day group was associated with the test substance in breast milk, indicating oral absorption and systemic exposure. The skin peeling resolved two weeks into the post-weaning recovery period. No skin peeling was observed in 0 (vehicle), 10 and 25 mg/kg bw/day groups and there was no test substance detected in the milk at these dose levels. Transient, but increased incidences of cold to touch occurred in the pups prior to weaning, and mild dehydration occurred on one or more occasions after weaning in both F1 male and female rats. Several F1 female rats also had observations of urine-stained abdominal fur during the post-weaning period. Based on the results of the study, the NOAEL for viability and growth in the offspring was set at 25 mg/kg bw/day. Treatment of dams with 500 mg/kg bw/day during the lactation period resulted in quantifiable levels of the test substance in the milk which was associated with increased incidences of skin peeling in the F1 generation pups. The clinical observation of skin peeling resolved by post-partum day 31, 2 weeks into the post-weaning recovery period.

 

Summary developmental toxicity: Developmental toxicity has been assessed in several studies a one-generation study and two developmental toxicity studies. 1) The key study is the one-generation study according to OECD 415 (0, 25, 100 and 500 mg/kg bw) in which the NOAEL of 25 mg/kg bw was found. In this study the animals were administered the test substance during gestation and lactation (OECD 415); 2a) In a second limit dose study (0 and 500 mg/kg bw) the exposure was again during gestation and lactation; 2b) In the same study with additional animals exposure (limit dose) during lactation was performed. 3) A study where pups were exposed during lactation only (0, 10, 25, 500 mg/kg bw).

In a one-generation reproductive toxicity study with rats, life birth index was significant lower and 6 females had a total litter loss. At 500 mg/kg bw a delay in onset and completion of the pinna unfolding was evident and less so at 100 mg/kg bw. Also the number of offspring passing surface righting, air righting and pupil reflex was reduced. Body weight and body weight gain of the pups was lower throughout lactation. Skin sloughing was seen in the pups at 500 and less so in 100 mg/kg bw. This is a rare effect and therefore additional studies were performed to see whether the effects was more pronounced when dosed during gestation or during lactation. In a limit dose study, a control and a dose of 500 mg/kg bw was administered. Again at 500 mg/kg bw significant maternal and development effects were seen. Pups of dams treated during gestation showed, however, less skin effects compared to dams treated during lactation. Therefore, another study was performed at which dams were only exposed during lactation at 500, 25 and 10 mg/kg bw. The test substance was found in the milk at 500 mg/kg bw but not in the 25 and 10 mg/kg bw. The skin sloughing was seen seen in the 500 mg/kg bw and was reversible in the post-weaning period at Day 35 of parturition. At this dose significant maternal toxicity was seen including liver toxicity possibly be indicative for overloading the metabolic pathway.

A NOAEL for parental toxicity of 25 mg/kg bw/day was established based on the OECD TG 415 being the key study for developmental toxicity, based on mortality, clinical signs, decreases in body weight gain and food consumption as well as liver effects at the highest dose of 500 mg/kg bw. In addition, at 500 mg/kg bw gestation length was increased and this wal also seen at 100 mg/kg bw but less pronounced. This gestation length increase at 100 mg/kg bw was finally the most critical effect on which base the NOAEL was set at 25 mg/kg bw. For the F1 generation, a NOAEL of 25 mg/kg bw/day was established based on delayed growth and on skin effects in male and female F1 animals. This NOAEL of 25 mg/kg bw was confirmed in the developmental toxicity in which the dams were exposure during lactation only.

Justification for classification or non-classification

Fertility: Based on the lack of adverse effects on fertility at the highest dose tested (500 mg/kg bw) in the one-generation study with rats, classification of the test substance for effects on fertility is not warranted according to EU CLP (EC 1272/2008 and its amendments).

Developmental toxicity: In the one-generation reproductive toxicity study with rats, administration of 500 mg/kg bw/day to dams resulted in decreased live birth indices and reduced pup viability, as well as reduced pup body weight and body weight gain. At 100 mg/kg bw increase in gestation length in the dams was followed by a delay in pup development (pinna unfolding, incisor eruption, eye opening and reflex development). This delay was minimal at 100 mg/kg bw and more severe at 500 mg/kg bw. The presence of skin peeling, acanthosis, and hyperkeratosis in the offspring were evident at dose levels of 100 and 500 mg/kg bw/day. These doses caused minimal and significant maternal toxicity, respectively. In addition, in two follow up studies these skin effects showed to be reversible during the post-weaning period. Therefore classification and labelling is not warranted according to EU CLP (EC 1272/2008 and its amendments).

Developmental toxicity via lactation: Pups are affected during lactation when dams are exposed at 500 mg/kg bw during lactation only. Some mortality was seen, some failed to thrive and body weight was lower until weaning. Also the skin peeling effects were seen at this dose and showed to be reversible during the post weaning period (Day Parturition 35). Marked maternal effects (body weight and liver) have been observed in repeated dose and reproductive toxicity studies. At this 500 mg/kg bw dose, the test substance was also detected in the mother's milk. At 25 mg/kg bw a NOAEL for maternal effects has been established and at this dose also no effects on pups are seen. The developmental effects including skin sloughing are much related to maternal toxicity and have shown to be reversible. Therefore, the test substance does not have to be classified for developmental toxicity or effects via lactation according to EU CLP (EC 1272/2008 and its amendments).