1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- Under the conditions of the test (Similar to OECD TG 421, GLP), no NOAEL for systemic toxicity could be derived. The LOAEL for systemic toxicity was determined to be 34 and 38 mg/kg bw in males and females, respectively. The fertility NOAEL was determined to be 121 and 134 mg/kg bw in males and females, respectively. The developmental NOAEL was determined to be 34 and 38 mg/kg bw in males and females, respectively.

- Under the conditions of the test (OECD TG 443, GLP), the systemic and fertility NOAEL was determined to be 94.1 and 91.7 mg/kg bw/day in males and females, respectively (taken into account the lowest HHCB intake prior to mating or during gestation and lactation).  The developmental NOAEL 123.2 and 95.2 mg/kg bw/day in males and females, respectively (taken into account the lowest HHCB intake during development of the pups).

- Under the conditions of the test (OECD TG 426, GLP), the systemic, fertility and developmental NOAEL were determined to be >=20 mg/kg bw.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

91.7 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The quality of the database is high because in different tests fertility has been investigated: in an OECD TG 426 study, in an simplified OECD TG 421 study, and in an OECD TG 443 study. These studies sufficiently adequately fulfill the REACH requirements.

Additional information

Toxicity to fertility has been investigated in three studies: in an simplified OECD TG 421 study, in an OECD TG 443 study and in an OECD TG 426 study.

DRF for EOGRT, CRL, 2020 (similar to OECD 421 but more limited design)

Method: A simplified OECD TG 421, oral diet study (in accordance with GLP) was carried out using 2 dose levels and historical controls were used as controls. The main objectives of this study were to evaluate the reproductive and developmental toxicity potential of the test item and to select dose levels for a subsequent Extended One-Generation Reproductive Toxicity study (OECD 443).

HHCB was administered continuously to groups of 10 male and 10 female Wistar Han rats at nominal doses of 690 and 2480 ppm for males and 305-690 ppm and 1090-2480 ppm for females corresponding to 34/38 and 121/134 mg/kg bw/day (male/female - taking the lowest intake of the test substance at each dose). Males were dosed for 29 days, i.e., 2 weeks prior to mating, during mating and up to the day of necropsy. Females were dosed during 2 weeks prior to mating, during mating, gestation and lactation and up to the day of necropsy, i.e., on Lactation Day (LD)21 to LD23 for females that delivered.

The following observations and examinations were performed: Mortality/morbidity, clinical signs, body weight, food consumption, estrous cycle determination, macroscopy at termination, organ weights and histopathology on kidney. In addition, the following reproduction/developmental parameters were determined: Mating and fertility index, precoital time, number of implantation sites, gestation duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality/morbidity, clinical signs, body weights, sex and macroscopy). In addition, the testes and epididymides were used for sperm analysis and microscopic examination.

Results: Parental Toxicity: Mortality: There was no unscheduled death in either group. Clinical signs: There was no test item-related clinical sign in either group for either sex. Body weight and food consumption: There was no test item-related effect on mean body weight or mean food consumption in any group for either sex. Organ weights when compared to historical controls: Males showed an increase in relative to body weight liver weights of 33% and 48% and females of 25% and 51%, at the low and high doses, respectively. Relative kidney weights were increased in males with 16% and 17% and in females 9% and 16% in the low and high doses, respectively. Relative thyroid weight was increased in males with 15% and 64% and in females with -4% and 26% at the low and high doses, respectively, versus HCD. Pathology findings were limited to increases in liver (both sexes), kidneys and thyroid glands (males) weights which were probably an adaptive effect enhanced by the test item and considered without toxicological relevance. In males only, there was also minimal to slight (690 ppm corresponding to 36 mg/kg bw/day) or slight to moderate (2480 ppm corresponding to 121 mg/kg bw/day) increased multifocal hyaline droplets characterized by eosinophilic cytoplasmic droplets in proximal convoluted tubules, noted in both treated groups indicating an exacerbation of normal pattern of droplets enhanced by the test item without degenerative or necrotic changes associated with nephropathy.

Fertility Toxicity: Length and regularity of the estrous cycle were not affected by treatment. There was no test item-related effect on mating performance or fertility. There was no test item-related effect on mean sperm counts, sperm morphology or motility, including the percentage of progressively motile sperm, in either group. There were 10/10 and 9/9 females that successfully completed delivery in the 38 mg/kg bw/day (305-690 ppm) and 134 mg/kg bw/day (1090-2480 ppm) groups, respectively. The mean duration of gestation was comparable in both groups (approximately 22 days). There was no test item-related effect on post implantation data in either group.

Developmental Toxicity: There was no test item-related effect on pup viability and litter sizes in either group. There was a lower percentage of male pups compared with female pups in both groups at birth when compared with the Historical Control Data (HCD) mean but remained within or close to the HCD range. There were no pup observations that suggested any association with the dosing of the dams. There was a dose-related lower mean pup body weight in both groups when compared with the HCD mean considered adverse at 134 mg/kg bw/day only in view of the magnitude of the change and because the difference compared to the HCD mean was consistent over time for both sexes.

Conclusion: Parental Toxicity:  Parental findings were limited to increases in liver (both sexes), kidneys and thyroid glands (males) weights (absence of macroscopic effects) which were could be an adaptive effect enhanced by the test item. For this study, the liver weight increase was considered a non-adverse adaptive response probably associated to hepatic hypertrophy. However, without histological examination of these organs a definitive conclusion cannot be drawn. There was also minimal to slight (dose of 690 ppm corresponding to 36 mg/kg bw/day) or slight to moderate (dose of 2480 ppm corresponding to 121 mg/kg bw/day) increased multifocal hyaline droplets for males characterized by eosinophilic cytoplasmic droplets in proximal convoluted tubules, noted only in males in both treated groups indicating an exacerbation of normal pattern of droplets enhanced by the test item without degenerative or necrotic changes associated with nephropathy. These effects need further characterisation before assigning them as a male rat-specific alpha 2u-globulin hydrocarbon nephropathy. Reproductive Toxicity: There was no sign of toxicity on reproduction at dose levels up to 121 mg/kg bw/day for males (2480 ppm) and 134 mg/kg bw/day for females (1090-2480 ppm). Developmental Toxicity: Developmental findings consisted of a dose-related lower mean pup body weight in both groups when compared with the HCD mean considered adverse at 134 mg/kg bw/day only in view of the magnitude of the change (up to 12% for males on PND1) and because the difference compared to the HCD mean was consistent over time for both sexes.

HHCB Dose selection for OECD TG 443 (to be presented in the Reproductive toxicity section)

Conclusion on dose selection:

Systemic toxicity: In a dietary 90-day and a dietary DRF for EOGRT study (simplified OECD TG 421In the repeated dose toxicity studies the relative liver weight (and thyroid) weight increased >=15% at 150 mg/kg bw indicating liver weight increase effects above the threshold. In an oral gavage developmental toxicity systemic effect in dams were seen on body weight and food consumption with a NOAEL of 50 mg/kg bw. These systemic effects were considered sufficient severe to present the highest dose of 150 mg/kg bw in the EOGRT.

Developmental toxicity was observed in pups in the DRF for EOGRT: Pup weights were decreased during lactation at 225 mg/kg bw to a toxic level (-12%). This decrease was a concern because this decrease may affect the fertility of the F1 and an F2 needed to be generated to complete the study. Therefore, dose levels for the OECD TG 443 are set to 150, 75 and 42.5 mg/kg bw.

 

Dose selection – Introduction

The dose selection for the DRF for the OECD TG 443 was based on the NOAEL in the 90-day dietary study: 150 mg/kg bw and the NOAEL for dams in the OECDTG 414 study: 50 mg/kg bw. Therefore, the dietary doses for the DRF (simplified OECD TG 421) were selected to be 225 and 62.5 mg/kg bw.

Dose selection – Effect data

Relative liver weight at 150 mg/kg bw in the 90-day study are exceeding +20% for males and are around +10% for females compared to control. Somewhat similar values are seen in the DRF at 225 mg/kg bw in which the females (+ 20.6% after ca. 54 days) compared to low dose values. For a sub-chronic study, a relative liver weight increase of around 10 – 15% may be considered adaptive in absence of other toxicological relevant findings in liver related enzymes (Interpretation of liver effects,

(https://webgate.ec.europa.eu/s-circabc/faces/jsp/extension/wai/navigation/container.jsp and select report interpretation of liver effects). An increase > 15% can be considered adverse.

Relative thyroid weights are similarly increased in at 150 mg/kg bw in the 90-day study: +20 and +11% for males and females, respectively.

In the DRF for the EOGRT, higher relative liver weight increases were seen. These were +43 and + 30% in males (28-days) and females (54 days), respectively, compared to the low dose of 62.5 mg/kg bw (control was not included). A systemic NOAEL could not be determined for this study based on these liver and thyroid weights in the absence of histopathological data. In the high dose of 150 mg/kg bw in the EOGRT test similar toxic effects are anticipated.

Fertility. No effects on fertility were seen and thus the dose selection is currently not critical for this endpoint.

Developmental toxicity. In the OECD TG 414, at 500 mg/kg bw, small but significant effects on body weight were seen in dams. Combined with other effects, the maternal NOAEL was set to 50 mg/kg bw. The pup weight was significantly decreased at 500 mg/kg bw, 7%, but minimally and not significant at 150 mg/kg bw: -2%. Developmental toxicity in the DRF for the OECD TG 443, at 225 mg/kg bw, there is a decrease in pup weight at day 1 (-12% and -10% for male and female pups, respectively, when compared with the HCD mean). This increases up to PND 4 and decreases from PND4 until weaning (see table 1). The pup weight decrease exceeding 10% may be a problem because an F2 generation is needed. Therefore, the 225 mg/kg bw is clearly too high. Only slight effects on pup weight are seen in the 62.5 mg/kg bw group. For pups the maximum dose would be somewhere between 62.5 and 150 mg/kg bw. To find some toxic effects a dose of 150 mg/kg bw is selected.

Table 1A: Results 90-day study

Dose levels (mg/kg bw)	Rel Liver wt (%)	Rel Thyroid wt (%)	Maternal BW effects
Male
5	+16	No effect
15	+5	+6
50	+16	+6
150	+21	+20
14d rec	+4	+15
Female
0	+5	-8
5	+5	+3	No effect
50	+5	+15	No effect
150	+10	+11	No effect
14d-rec	+4	+13	No effect

 

Table 1B: Results OECD 414

Dose levels (mg/kg bw)	Maternal BW effects (%)	Prenatal pup weight
0	391.9g (Day 20)	3.48 g
50	-2.3*	-
150	-3.3*	-2
500	-5*	-7

*slight but significant

 

Table 1C: Results DRF for 443 diet

Dose levels (mg/kg bw)	Rel Liver wt (%)	Rel Thyroid wt (%)	Maternal BW effects (%)	Pup wt Day 1 (%)	Pup wt Day 4 (%)	Pup wt Day 21 (%)
Males
62.5				-9 (vs HCD)	-9.5 (vs HCD)	-5 (vs HCD)
225	+11	+43		-12 (vs HCD)	-15 (vs HCD)	-11 (vs HCD)
Females
62.5	Used as control	Used as control	Used as control 420 g	-9 (vs HCD)	-9.5 (vs HCD)	-4 (vs HCD)
225	+20.6	+30	-4	-10 (vs HCD)	-13 (vs HCD)	-9 (vs HCD)

HCD=historical control

EOGRT according to OECD 443, CRL, 2021

Method: F0-generation: The test item was administered by the oral route (diet admixture) continuously to groups of 25 male and 25 female Wistar rats of the F0 generation at nominal doses of 470, 825 and 1650 ppm (adapted during the lactation period to the higher body weight/food consumption ratio). F0 males were dosed for 10 weeks prior to mating, during mating and up to the day before necropsy. F0 females were dosed during 10 weeks prior to mating, during mating, gestation and lactation and up to the day before necropsy, i.e., on Lactation Day (LD) 21 to LD23 for females that delivered. A fourth group received the control item (basic powdered diet) under the same conditions. The inseminated females were allowed to litter and development of the offspring was observed up to weaning on Postnatal Day (PND) 21. The following parameters and endpoints were evaluated in all F0 animals: Mortality, clinical signs, body weights and body weight gains, food consumption, clinical pathology, thyroid hormone levels, estrous cycles, mating performance and fertility, delivery and litter data, sperm quality, macroscopic observations, organ weights and histology findings. F0 males were euthanised after at least 18 weeks of exposure and F0 females after at least 17 weeks of exposure.

Method: F1 generation: From weaning, the selected F1-pups were allocated to 3 cohorts (1A, 1B and 1C) of 20 males and 20 females each. F1 animals were exposed to the test item by the oral route (diet admixture) continuously from PND21 up to the day before or the day of scheduled necropsy at the same nominal doses of 470, 825 and 1650 ppm (adapted during the lactation period to the higher body weight/food consumption ratio). A fourth group received the control item (basic powdered diet) under the same conditions. F1 animals were observed for mortality, clinical signs, body weights and body weight gains, food consumption, clinical pathology (Cohort 1A), thyroid hormone levels (PND4 pups and Cohort 1A), pup anogenital distance, pup number of aerola/nipples, estrous cycles (Cohort 1A), sexual maturation, sperm quality (Cohort 1A), macroscopic observations, organ weights and histology findings. Additionally, spleen samples were taken for splenic lymphocyte subpopulation analysis from 10 rats/sex/group from F1 Cohort 1A animals. F1 animals were sacrificed after sexual maturation for Cohort 1C or between PND85 and PND93 for Cohort 1A. Surplus offspring (10/sex/group) not included in any of the F1 cohorts, were selected for thyroid hormone analysis and organ weights on PND21. F1 females from Cohort 1B were mated and allowed to litter and development of the offspring was observed up to weaning on PND21. The following reproduction/developmental parameters were determined: Mating performance and fertility, and delivery and litter data. For Cohort 1B, F1 males were sacrificed after at least 20 weeks of exposure and F1 females after at least 19 weeks of exposure.

Method: F1 generation: F2 offspring (from Cohort 1B) were observed for early postnatal development, mortality, clinical signs, body weights, sex, anogenital distance, number of aerola/nipples, organ weights and macroscopy. F2 offspring were sacrificed on PND21-23.

Results: Samples collected from formulations at nominal concentrations of 470, 825 and 1650 ppm on Day 1, Week 11 and Week 22 of treatment and from nominal concentrations of 205 and 1650 ppm on Week 16 and on Week 32 were accurate and homogeneous. No test item was present in the vehicle sample. Samples collected at the rodent unit at concentration of 205 and 1650 ppm were stable after 4 days refrigerated or at room temperature but a loss of 19.9% and 25.1% of the test item in the diet was observed when formulations were stored for 8 days at room temperature. This was consistent with the overestimation of the dose levels (+25%) when compared with the target doses.

Results: F0 Generation: There were no test item-related death in any group for F0 animals. There was no test item-related clinical sign in any group for F0 animals. There was no test item-related effect in any group in the mean F0 body weight gain or food consumption for either sex with the exception of a slightly lower mean body weight gain for females during the gestation period at 1650 ppm considered related to the lower mean pup weight in this group. There was no test item-related effect on TSH serum concentration either sex or on total T4 serum concentration for females.

Necropsy: At necropsy of F0 animals, bilateral diffuse follicular cell hypertrophy of the thyroid gland was observed in males and females at all doses and a minor dose-related decrease in mean total T4 serum concentration for males that remained within the HCD range. This finding was dose-related and was minimal or mild, correlated with an increased organ weight and was considered not adverse in view of the low magnitude of the change. There was no test item-related macroscopic in any group for either sex.

Fertility: There was no test item-related effect in any group on F0 male and female mating and fertility, male copulation, mean number of implantations, mean estrous cycle lengths, mean precoital intervals, gestation lengths and sperm parameters. There were no test item-related effects on hematology, coagulation, serum clinical chemistry and urinary parameters for F0 animals at any dose for either sex.

Results: F1 Generation: Pups: There was no test item-related effect on F1 postnatal survival in any group. The mean number of pups delivered was comparable with that of the control group and the historical control data mean in all treated groups. There were no test item-related effects on F1-pup clinical condition, anogenital distance, or areolae/nipple at any dose level. There was a dose-related lower mean pup body weight in all groups when compared with the control group and the HCD mean on PND1 which was persistent throughout the lactation period at 1650 ppm. This finding was considered not adverse at 1650 ppm since the mean pup values at 1650 ppm remained within or very close to the HCD range on PND21. In addition, there was no consistency with respect to the lower mean pup body weight on PND1 with the second generation. There were no test item-related effects on T4 and TSH levels in F1-pups on PND4 and PND21.

Adults: There was no test item-related effect on the mean ages at attainment of balanopreputial cleavage and vaginal opening for F1 animals in any group. The duration from vaginal opening to first estrous in the treated groups was comparable to the control group. There was no test item-related clinical sign in any group for either sex. There was a dose-related effect in all groups in the mean body weight gain prior to mating (males and females) and during the gestation period (Cohort 1B) associated with a slight reduction in mean food consumption considered not toxicologically significant in view of the low magnitude of the changes and considering that the terminal mean body weight for males at 1650 ppm on Day 120 was comparable with the HCD. There were no test item-related effects on hematology, coagulation, serum clinical chemistry and urinary parameters for the F1 males and females after approximately 13 weeks of dosing (Cohort 1A) at any dose. There was no test item-related effect on the splenic lymphocyte population for the F1 males and females after approximately 13 weeks of dosing (Cohort 1A) at any dose.

Necropsy: At necropsy, higher mean absolute and relative thyroid weights were noted for male and female groups (Cohorts 1A and 1B) corelated with a minor increase in TSH level for females at 1650 ppm and a slight dose-related decrease in T4 levels for males when compared with the concurrent control. A relationship to HHCB was likely at 1650 ppm for males but considered not adverse in view of the low magnitude of thyroid weight increase, where it correlated with follicular cell hypertrophy. For females, there was no histopathologic correlate in the thyroid gland, and a relationship to HHCB administration was therefore considered equivocal. In the liver, higher mean relative organ weights were noted in male given 1650 ppm from both Cohorts 1A and 1B considered not adverse in view of the low magnitude of the change and in the absence of histology findings. There were no test item-related macroscopic or other microscopic findings and no other difference in organ weights for F1 animals at any dose level. There was no difference in TSH serum levels at 470 and 825 ppm or in T4 serum levels in any group for females or in TSH serum levels for males in any group.

Fertility: There was no test item-related effect in any group on F1 males and females mating and fertility, male copulation, mean number of implantations, mean precoital intervals, gestation lengths (Cohort 1B). There were no test item-related effects on mean F1 estrous cycle lengths or spermatogenic parameters noted in any group (Cohort 1A).

F2 Generation: There was no test item-related effect on F2 postnatal survival in any group and no test item-related effect on the mean number of pups delivered. There were no test item-related effects on F2-pup clinical condition, anogenital distance, or areolae/nipple at any dose level. There was a dose-related lower mean pup body weight at 825 and 1650 ppm for both sexes when compared with the control group and the HCD mean. This finding was considered not adverse given that there were no differences in mean pup body weight on PND1 and that mean pup values on PND21 at 825 and 1650 ppm remained within or very close to the HCD range. Necropsy: At necropsy of F2 animals, findings were limited to a test item-related increase liver weight for males and females at 1650 ppm considered not adverse in view of the low magnitude of the change. There was no macroscopic finding in any group.

Conclusion: A parental and reproduction NOAEL of at least 1650 ppm was derived, corresponding to a minimum achieved intake of 94.1 and 91.7 mg/kg/day in males and females, respectively (taken into account the lowest HHCB intake prior to mating or during gestation and lactation). Test item-related preweaning developmental effects consisted in a dose-related lower mean pup body weight in all groups from PND1 for the F1 generation or at 825 and 1650 ppm on PND21 for both sexes for the F2 generation. This finding was considered not adverse as all mean values on PND21 for both generations remained within or very close to the HCD range. Postweaning developmental effects included a dose-related effect in all groups on mean body weight gain prior to mating (males and females) and during the gestation period (Cohort 1B) associated with a slight reduction in mean food consumption considered not toxicologically significant in view of the low magnitude of the changes and given that terminal mean body weight for males was comparable with the HCD. In addition, the minor differences in mean pup body weight or postweaning mean body weight gains were not associated with any other test item-related developmental effect. A developmental NOAEL of at least 1650 ppm was derived, corresponding to a minimum achieved intake of 123.2 and 95.2 mg/kg/day in males and females, respectively (taken into account the lowest HHCB intake during development of the pups).

Peri/postnatal Developmental Neurotoxicity test similar to OECD TG 426, 1996

Method: In a study (similar to OECD TG 426, in accordance with GLP) designed to determine the effects of test material (purity >95%) on the neonate when exposed through nursing, test material was administered at dosages of 0, 2, 6 or 20 mg/kg bw/day once daily by gavage in corn oil to groups of 28 time-mated rats (Crl:CD BR VAF/Plus strain) from day 14 of pregnancy (end of organogenesis) through to weaning on day 21 post partum. The females were allowed to litter and rear their young to weaning. From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capacity. The F1 generation was only exposed to HHCB in utero during the perinatal phase and through transfer in the milk of the lactating dams. Actual intakes cannot be determined because milk consumption during nursing was not measured. After parturition, the young were counted, sexed, weighed and examined for external abnormalities. On day 4 post partum the pups were weighed and all litters containing more than 8 pups were culled to 8 retaining, where possible 4 males and 4 females. During the preweaning period, all pups were examined to determine the age of reaching certain developmental stages by examining surface righting reflex, startle reflex, air righting reflex and pupil reflex. This F1 generation was also evaluated for behavioural effects by examining changes in motor coordination and balance, activity and avoidance. When the F1 generation reached approximately 84 days of age (having been continuously observed for signs of adverse health) they were mated one male to one female avoiding brother-sister pairings. The females were examined before and after mating to determine time of pregnancy, marked anomalies of the oestrous cycle, median pre-coital time, whether pregnancy had occurred and terminated and duration of pregnancy. The offspring (F2 generation) were examined for abnormalities at parturition and periodically until day 21 post partum at which time the study was terminated.

Results: There were no effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the F1 generation during the late prenatal phase, or on postnatal growth, no changes in post weaning behavioural tests or mating performance were seen and post mortem examination of F1 males and females, reproductive capacity, litter data and macroscopic post mortem examination of F2 pups did not reveal abnormalities.

Conclusion: The highest dose administered; 20 mg/kg bw/day produced no adverse effects. Under the conditions of the test, the systemic NOAEL was determined to be >20 mg/kg bw. The fertility NOAEL is >= 20 mg/kg bw and the developmental NOAEL is >= 20 mg/kg bw.

Effects on developmental toxicity

Description of key information

- Under the conditions of the test (OECD TG 421, GLP, Rat), the maternal LOAEL was determined to be 34 mg/kg bw/day. The developmental NOAEL was determined to be 34 mg/kg bw/day.

- Under the conditions of the test (OECD TG 443, GLP, Rat), the maternal NOAEL was determined to be >91.7 mg/kg bw/day. The developmental NOAEL was determined to be >95.2 mg/kg bw/day.

- Under the conditions of the test (OECD TG 426, GLP, Rat), the maternal NOAEL was determined to be >20 mg/kg bw/day. The developmental NOAEL was determined to be >20 mg/kg bw/day.

- Under the conditions of the test (OECD TG 414, GLP, Rat), the maternal NOAEL was determined to be 50 mg/kg bw/day. The developmental NOAEL was determined to be 150 mg/kg bw/day.

- Under the conditions of the test (OECD TG 414, GLP, Rabbit), the maternal NOAEL was determined to be 30 mg/kg bw/day. The developmental NOAEL was determined to be >100 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The quality of the database if high because in different tests developmental toxicity has been investigated: in a simplified OECD 421 study, in an OECD 443 study, in a study in accordance with OECD 426, in an OECD 414 study in rats and in an OECD 414 study in rabbits. These studies sufficiently adequately fulfill the REACH requirements.

Additional information

Developmental toxicity has been investigated in five studies: in an simplified OECD TG 421 study, in an OECD TG 443 study, in an OECD TG 426 study in an OECD TG 414 study using rats and in an OECD TG study using rabbits.

Only the two developmental toxicity studies according to OECD TG 414 are presented in the present section. The developmental toxicity in the combined reproductive toxicity studies (similar to OECD 421, 443 and 426) are fully presented in the reproductive/fertility section.

OECD 414: DRF: Rat, RIFM, 1997

A dosage range finding study in accordance with GLP was conducted to provide information for the selection of dosages to be used in a developmental study. In this study, groups of 8 pregnant Sprague-Dawley rats were administered HHCB in corn oil by gavage (5 ml/kg bw/day) at doses of 100, 250, 500 or 1000 mg/kg bw/day on days 7 through 17 of pregnancy. A control group of 19 pregnant rats received corn oil only. Dams on all dose-levels showed reduced body weight gain during the treatment period (day 7-18 of gestation). Dams on 500 and 1000 mg/kg bw/day showed reduced body weight gain during treatment plus post treatment period (day 7-20 of gestation). Reduced absolute and relative food consumption was seen in dams of all treated groups during treatment period (day 7-18) and during treatment plus post treatment period (day 7-20). Mean foetal body weights were 89.3% of controls at 1000 mg/kg bw/day. No other treatment related effects were seen.

Based on the results of this study, dosages of 0 (Vehicle), 50, 150 and 500 mg/kg bw/day of test item were selected for the developmental toxicity study in rats.

 

OECD 414: Main study: Rat, RIFM, 1997

Method: In a developmental toxicity study (OECD TG 414, GLP) the test substance in corn oil was administered by gavage to groups of 25 female Sprague-Dawley rats on days 7 through 17 of presumed gestation at dosages of 0, 50, 150 and 500 mg/kg bw/day. The dams were observed for signs of toxicity and body weights and feed intake were recorded. On day 20 of gestation, the dams were sacrificed, and gross necropsy was performed. The number of corpora lutea in the ovaries was recorded and the uteri were examined for pregnancy, number and distribution of implantations, live and dead foetuses and early and late resorptions and the placenta were examined. All foetuses were weighed and examined for sex and gross external abnormalities. One half of the foetuses in each litter were examined for soft tissue alterations. The remaining foetuses were examined for skeletal alterations.

Results, Clinical signs: No mortality was noted. The 500 mg/kg bw/day dosage group had four to nine (p ≤ 0.01) rats with excess salivation (9 animals), urine-stained abdominal fur (7 animals), red or brown substance on the forepaws (4 animals) and alopecia (6 animals). Body weights: Maternal body weights were unaffected by the 50 mg/kg bw/day dosage group. The 150 and 500 mg/kg bw dosage groups had statistically significant dose-dependent reductions in maternal body weight gains for the entire dosage period (day 7 to 18). For the entire interval after initiation of treatment, (day 7 to 20) maternal body weights were reduced and significantly reduced in the 150 and 500 mg.kg bw/day dosage groups, respectively. Food consumption: Absolute and relative feed consumption values were unaffected by the 50 mg/kg bw dosage group. Absolute and relative feed consumption values for the entire dosage period were reduced in the 150 and 500 mg/kg bw dosage group after initiation of dosage and in the highest group for the entire gestation period.

Fertility: Pregnancy occurred in 21 to 25 female rats in each dosage group. There were 25, 24,24 and 21 pregnant dams Caesarean-sectioned on DG 20 in the 0 (vehicle). 50, 150 and 500 mg/kg bw day dosage group, respectively. No abortions or premature deliveries occurred during the study.

Developmental: Foetuses in the 500 mg/kg bw/day dosage group showed significantly reduced body weights, considered test item related. Significant increases in the litter and foetal incidences of skeleton (vertebral/rib) malformations were found in the 500 mg/kg bw and considered to be associated with administration of this dosage of the test article to the dams. Malformations occurring in foetuses of dams administered dosages less than 500 mg/kg bw day were considered unrelated to the test article because the severity of the expression of the malformation was not consistently dose dependent. There were no dosage-dependent, or statistically significant differences in the litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early and late resorptions, percent resorbed conceptuses and percent live male/female foetuses were comparable among the four dosage groups and did not differ significantly. No dam had a litter consisting of only resorbed conceptuses and there were no dead foetuses.

Conclusion: Based on a reduction in adverse clinical observations (500 mg/kg bw/day group only), maternal body weight gains and feed consumption values for the dosing period (days 7 to 18 of gestation), the maternal NOAEL was determined to be 50 mg/kg bw/day. Based on a reduction in foetal body weight, increased incidences of foetal-skeletal (vertebral/rib) malformations, and decreased ossification of sternal centra and metatarsals seen at 500 mg/kg bw/day, the developmental NOAEL was determined to be 150 mg/kg bw/day. The test substance was not selectively toxic to development because adverse effects on development occurred only at dosages that produced toxic effects (adverse clinical observations, decreased body weight and feed consumption values) in the dams.

OECD 414: DRF and MTD study: Rabbit, CRL, 2020

The dose levels of the main test were selected based on a dose-range finding prenatal developmental toxicity study (DRF-study) in the rabbit and a tolerability study in the non-pregnant rabbit (MTD-study).

DRF-study: In the DRF-study, 6 pregnant New Zealand White rabbits were dosed at 30, 100 and 300 mg/kg/day with HHCB in corn oil at a dose volume of 4 mL/kg from GD6 to GD9 and 2 mL/kg from GD10 to GD28. Severe vehicle-related maternal toxicity was noted including negligible food consumption leading to body weight loss in all groups, including the control, that led to the death or  premature euthanasia of 1, 2, 3 and 3 females in the control, 30, 100 and 300 mg/kg/day groups, respectively. However, there was no clear test item-related embryo-fetal toxicity in any group.

MTD-study: In the MTD-study, non-pregnant New Zealand White rabbits were dosed at 300 mg/kg/day with HHCB in corn oil at 1.25 mL/kg during 8 days and was associated with negligible food consumption for all animals and was therefore above the maximum tolerated dose. Animals given corn oil only at 1.25 mg/mL also had slightly lower food consumption when compared with the pre-test period. Following a recovery period, the animals received a dose of 100 mg/kg/day in corn oil at 0.8 mL/kg for 7 days and was associated with tolerable effects on mean food consumption and body weight gain. 

Conclusion: Based on these findings, the doses of 10, 30 and 100 mg/kg/day were considered appropriate for this prenatal developmental toxicity study in the pregnant rabbit with the high dose expected to result in tolerable maternal effects.

 

OECD 414: Main study: Rabbit, CRL, 2020

Method: The effect of HHCB on prenatal development in the rabbit following administration from implantation to the day before caesarean section (from Gestation Day (GD)6 to GD28) was investigated. New Zealand White rabbits were exposed to 0 (Corn oil), 10, 30, and 100 mg/kg bw in a OECD TG 414 study following GLP. The following parameters and endpoints were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, maternal gross necropsy findings, litter parameters and fetal morphology.

Results, Formulation Analysis: All analyzed formulations samples prepared at nominal concentrations of 12.5, 37.5 and 125 mg/mL of HHCB in vehicle (corn oil), taken from each preparation, including the vehicle, on GD6 were in agreement with acceptance criteria for accuracy and homogeneity and no test item was present in the vehicle sample.

Mortality: One female at 100 mg/kg/day aborted after developing a progressive reduction in food consumption and reduced faecal output from GD6 that became negligible from GD12 and led to 25% of body weight loss from GD12 to GD27. This finding in the high dose group was likely due to an exacerbation of the vehicle-related effects by the test item. One and two females in the 30 and 100 mg/kg/day groups, respectively, were preliminary euthanized (for ethical reason or abortion) after occurring marked reduction in mean food consumption leading to body weight loss that started from the arrival of the animals. These preliminary euthanasia's could not be attributed to the test item or the vehicle in view of the difficult acclimatization observed for both animals. Finally, 1 female at 100 mg/kg/day was found dead just after dosing on GD7 and considered related to the administration procedure (misgavage) and not test item related.

Clinical signs: Vehicle-related clinical signs were noted at comparable incidences between control and treated groups, including reduced faecal output as well as pale or soft/small faeces and absent urine.

Body weight / Food consumption: There was a markedly reduced mean food consumption during the overall dosing period (GD6 to GD29) in all groups including control when compared with the historical control data resulting in a marked reduction in mean body weight gain in all groups. However, the effect was more pronounced for females treated at 100 mg/kg/day when compared with the control group.

Necropsy Findings: There were no test item-related macroscopic finding.

Gravid Uterus: There was no test item-related effect on mean gravid uterus weight in any group.

Litter Data: There were 21, 18, 16 and 17 pregnant females at terminal caesarean in the control, 10, 30 and 100 mg/kg/day groups, respectively. All of these females had viable fetuses with the exception of 1 given 30 mg/kg/day. Pre-implantation data were comparable within all groups and there was no test item-related effect on post-implantation data in any group. The mean number of implantation sites and the mean live litter size in treated groups were comparable or higher than those of the control group and/or historical control data mean. Pup weight: There was a, equivalent lower mean fetal weight in all groups, including control group, when compared with the historical control data range. This was considered to be related to the marked vehicle-related maternal toxicity rather than a test item-related effect. There was no test item-related effect on fetal sex ratio in any group.

Fetal Examinations: There were no external, visceral or skeletal malformations that were considered to be test item-related and the incidence of other visceral or skeletal variations did not suggest any association with the test item.

Conclusion: In conclusion, daily oral (gavage) administration of HHCB at doses of 10, 30 and 100 mg/kg/day in corn oil in the pregnant New Zealand White rabbit from GD6 to GD28 was associated with a marked reduction in food consumption and reduced faecal output leading to a markedly lower mean body weight gain in all groups when compared with the historical control data. These findings noted amongst the treated and control groups were indicative of vehicle-related maternal toxicity. Although females in the control group appeared more sensitive to the vehicle-related maternal toxicity when compared with the 10 and 30 mg/kg/day groups, the effects on mean body weight gain and food consumption were more pronounced at 100 mg/kg/day (-11% and -5%, respectively) when compared with the concurrent control group. This difference was indicative of a test item-related exacerbation of the vehicle-related effects. In addition, 1 female at 100 mg/kg/day aborted after incurring a progressively more severe reduction in food consumption that led to a body weight loss of -25% compared with no similar cases in other groups. A NOAEL for test item-related maternal toxicity was therefore set at 30 mg/kg/day. There was no test item-related embryo-fetal toxicity in any group. However, consistent with the severity of the vehicle-related maternal toxicity, mean fetal weight was lower in all groups, including the control, when compared with the historical control data but considered not test item-related as there was no difference between the treated and control groups. The NOAEL for prenatal development was 100 mg/kg/day.

Justification for classification or non-classification

Based on the results of the available reproductive and developmental toxicity studies the substance does not have to be classified for reproductive toxicity (fertility and developmental toxicity) according to EU CLP (EC 1272/2008 and its amendments).