5H-Cyclopenta[h]quinazoline, 6,7,8,9-tetrahydro-7,7,8,9,9-pentamethyl-

Endpoint summary

Description of key information

- The dietary repeated dose toxicity NOAEL is set to 9.1 mg/kg bw based on a Reproscreen study with additional repeated dose toxicity parameters (OECD TG 421)

- The LOAEL is 43 mg/kg bw based on the dietary repeated dose toxicity study according to OECD TG 407

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

9.1 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

-  The repeated dose effects could be related to the liver-thyroid-pituitary axis: Increase in liver metabolism, decrease T4, increase in TSH by pituitary and increase in thyroid excretion and therefore thyroid hypertrophy.

- Contra-intuitive are the ‘side’ effects: in case the pituitary would be stimulated you would expect increase in weight, while decrease is seen in males and females, without a clear dose relation. There is hypertrophy (without weight increase). Hypertrophy may be a secondary result of gonad (and thyroid?) disfunction. In addition, the effect of degranulation (decrease in hormone secretion?) is not clear yet. No degranulation is seen in females. (https://focusontoxpath.com/pathology-of-the-mouse-pituitary-gland/

- In the thyroid there is hypertrophy but not weight increase, while thyroid (relative) weight and hypertrophy are usually a combined effect.

- The male and female reproductive organs are affected prostate and uterus weight decrease at 40 mg/kg bw and 67 mg/kg bw, respectively. It is not clear whether this causes the pituitary changes or are a result of this.

Additional information

The systemic toxicity of the substance is derived from 3 studies: An oral gavage DRF study a dietary OECD TG 407 and dietary Reproscreen OECD TG 421 study. In the OECD TG 407 only a LOAEL could be derived, therefore in the OECD TG 421 critical organ toxicity parameters were included to find a systemic NOAEL, which was the case. Therefore this latter test is presented first. 

Reproductive Toxicity Screening study (OECD TG 421) with number of systemic toxicity parameters 

The substance was administered by continuous dietary admixture to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up 28 days, at dietary concentrations of 50, 150 and 500 ppm (equivalent to a mean achieved dosage of 3.0, 9.1 and 31.0 mg/kg bw/day for males and 3.4, 10.5 and 34.0 mg/kg bw/day for females). A control group of twelve males and twelve females was fed basal laboratory diet. This study was conducted according to OECD TG 421 and GLP principles. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results, mortality and clinical signs: showed that there were no unscheduled deaths. There were no clinical signs apparent for animals of either sex treated with 50, 150 or 500 ppm. There were no treatment-related effects detected in body weight development for males treated with 50, 150 or 500 ppm. Females treated with 500 ppm showed a reduction in body weight gain during maturation, the first two weeks of gestation and during lactation. No toxicologically significant effects were detected in females treated with 150 or 50 ppm. There were no treatment-related effects on food consumption or food conversion efficiency for males treated with 50, 150 or 500 ppm. Females treated with 500 ppm showed a slight reduction in food consumption and food conversion efficiency during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 150 or 50 ppm. There were no treatment related effects on water consumption.

Organ related effects: Animals of either sex treated with 500 ppm and females treated with 150 ppm showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Females treated with 500 ppm also showed a statistically significant increase in thyroid/parathyroid weight and reduced pituitary weight both absolute and relative to terminal body weight. No such effects were detected in males treated with 150 ppm or in animals of either sex treated with 50 ppm. There were no microscopic abnormalities detected.

Conclusion: Based on these findings, the oral administration of the test substance to rats at dietary concentrations of 50, 150 and 500 ppm resulted in a reduced body weight gain, reduced initial food consumption, increased thyroid weights and reduced pituitary weights in females treated with 500 ppm and increased liver weights in animals of either sex treated with 500 ppm and in females treated with 150 ppm. The systemic toxicity in the present study results in a NOAEL of 150 ppm, equivalent to 9.1 mg/kg bw. 

 

Dose range finding study for the Repeated Dose Toxicity study (2013)

The study was designed to provide information for further repeated dose toxicity studies. The substance was administered by gavage to three groups, each of three male and three female Wistar Han:RccHan:WIST strain rats, for up to seven consecutive days, at dose levels of 50, 100 and 175 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.

Results: One female treated with 175 mg/kg bw/day was killed in extremis on day 3. The remaining animals of either sex from this treatment group were also terminated on day 3. There were no further unscheduled deaths. All females treated with 175 mg/kg bw/day had hunched posture on day 3. The female that was killed in extremis also had tip toe gait, a decreased respiratory rate, piloerection and lethargy. No clinical signs of toxicity were detected in males treated with 175 mg/kg bw/day or animals of either sex treated with 100 or 50 mg/kg bw/day. All animals of either sex treated with 175 mg/kg bw/day and females treated with 100 mg/kg bw/day showed actual body weight losses on day 3. A reduction in body weight gain was also evident in 100 mg/kg bw/day males on Day 3. No adverse effect on body weight gain was detected in animals of either sex treated with 50 mg/kg bw/day. Animals of either sex treated with 175 mg/kg bw/day showed a substantial reduction in food consumption on Day 3. A reduction in food consumption was also evident in males treated with 100 mg/kg bw/day between days 1 and 3 and between days 1 and 5 for females treated with 100 mg/kg bw/day. No adverse effect on food consumption or food efficiency was detected in animals of either sex treated with 50 mg/kg bw/day. Males treated with 175 mg/kg bw/day showed an increase in water consumption between days 1 and 3. Females treated with 100 mg/kg bw/day showed an increase in overall water consumption when compared to control females. No adverse effect on water consumption was detected in females treated with 175 mg/kg bw/day, males treated with 100 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day. Macroscopy: All males treated with 175 mg/kg bw/day had a mottled liver, a small spleen, a distended stomach and a thin non-glandular region of the stomach. The non-glandular region of the stomach was also reddened in two of these males. All females treated with 175 mg/kg bw/day had a discoloured liver, a distended stomach and a reddened and thin non-glandular region of the stomach. One female also had a small spleen and another female also had a pale glandular region of the stomach. At 100 mg/kg bw/day, one female had sloughing on the non-glandular region of the stomach and the remaining two females had pale foci on the non-glandular region of the stomach. No macroscopic abnormalities were detected in males treated with 100 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day. Animals of either sex treated with 100 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight when compared to control animals. Females treated with 50 mg/kg bw/day also showed an increase in absolute and relative liver weight. No such effects were detected in males treated with 50 mg/kg bw/day. In conclusion, the oral administration of the substance to rats by gavage, at dose levels of 50, 100 and 175 mg/kg bw/day, resulted in treatment related effects in animals of either sex treated with 175 and 100 mg/kg bw/day. At 50 mg/kg bw/day, the only treatment related effect detected was an increase in female absolute and relative liver weight.

Repeated dose toxicity study (OECD 407); Key study

In a 28-days repeated dose toxicity study ( according to OECD TG 407 and GLP principles) with a 2-week recovery period, the test substance wasby continuous dietary admixture to three groups, each of five male and five female WistarHan™:RccHan™:WIST strain rats. The rats were exposed to dietary concentrations of 500, 800 and 1200 ppm (equivalent to a mean achieved dosage of 43.1, 67.6 and 101.0 mg/kg bw/day for males and 47.4, 58.0, 85.8 mg/kg bw/day for females respectively). A control group of five males and five females were treated with basal laboratory diet. Two recovery groups, each of five males and five females, were treated with the high dose (1200 ppm) or basal laboratory diet for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, body weight change, food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination including organ weights and histopathological examination of selected tissues was performed.

Results, mortality and clinical signs showed that there were no treatment related deaths. One male treated with 500 ppm died during the blood sampling procedure on say 28 of the study. There were no clinical signs for this animal prior to the death or any macroscopic findings at necropsy. In the absence of any other unscheduled deaths in this study, this finding was likely caused by a heightened stress response associated with the blood sampling procedure and was considered unrelated to treatment. There were no clinical signs for any of the animals throughout the study. There were no treatment-related changes in the behavioural parameters at 500, 800 and 1200 ppm. There were no toxicologically significant changes in functional performance at 500, 800 and 1200 ppm. There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 500, 800 and 1200 ppm. During the first week of the treatment, group mean body weight gains for animals of either sex from all treatment groups were reduced when compared to controls. Body weight gain for animals of either sex treated with 1200 ppm remained lower during weeks 2 and 4 and during week 3 for females. Animals of either sex treated with 800 ppm also showed a reduction in body weight gain during the final week of treatment. During the treatment-free period, body weight development for the high dose males was similar to controls and for females, was higher than controls. Males treated with 1200 ppm showed a reduction in food consumption during the first week of treatment. Females from this treatment group showed a reduction in food consumption throughout the treatment period. Females treated with 800 ppm showed a reduction in food consumption during the first and last week of treatment. Food efficiency was reduced in animals of either sex from all treatment groups during the first week of treatment. Females treated with 1200 ppm continued to show reduced food efficiency during week 2 whilst males treated with 1200 and 800 ppm showed a reduction in food efficiency during the final week of treatment. No such effects in food consumption were detected in males treated with 800 ppm or in animals of either sex treated with 500 ppm. No treatment-related adverse effects on water intake were observed. Fertility: Two females treated with 1200 ppm and one female treated with 800 ppm were acyclic throughout the assessment period. The remaining females treated with 1200 and 800 ppm, all 500 ppm females and all recovery females showed evidence of oestrus. Males from all treatment groups showed an increase in prothrombin time at the end of the treatment period. No toxicologically significant effects were detected in females from all treatment groups or in recovery animals of either sex. Biochemistry: At the end of the treatment period, animal of either sex from all treatment groups showed a statistically significant increase in gamma glutamyl transpeptidase. Females from all treatment groups also showed a statistically significant increase in cholesterol and phosphorus and a statistically significant reduction in albumin/globulin ratio. Males from all treatment groups showed a statistically significant reduction in bilirubin. Males treated with 1200 ppm also showed a statistically significant reduction in urea, calcium concentration and alkaline phosphatase and a statistically significant increase in phosphorus. The effect on urea, calcium concentration and alkaline phosphatase also extended to males treated with 800 ppm. Urinalysis: There were no toxicologically significant effects detected in the urinalytical parameters examined. Organ effects: One female treated with 800 ppm had small adrenals and a small uterus and cervix. No such effects were detected in any other treated female. Macroscopic necropsy findings of males did not indicate any adverse effect of dietary exposure at 500, 800 or 1200 ppm. Males treated with 1200 ppm showed a reduction in sperm concentration and motility values. There were no treatment-related effects detected in morphological assessments or in homogenisation-resistant spermatid counts. No such effects in sperm concentration or motility values were detected in males treated with 800 or 500 ppm. At the end of the treatment period, animals of either sex from all treatment groups showed an increase in liver weight and a reduction in pituitary weight both absolute and relative to terminal body weight. Males from all treatment groups also showed a reduction in prostate and seminal vesicle weight both absolute and relative to terminal body weight. Females treated with 1200 and 800 ppm also showed a reduction in absolute and relative uterus and cervix weight. A reduction in absolute and relative thyroid weight was evident however this was confined to females treated with 800 and 500 ppm females only. At the end of the treatment free period, females continued to show an increase in liver weight both absolute and relative to terminal body weight. Microscopic abnormalities were detected in the liver where Centrilobular hypertrophy was present in the liver of animals of either sex from all treated groups, in a dose dependant pattern. This change resolved after the recovery period. Periportal vacuolation (fat-type mainly macro vesicular) was present in animals of either sex from all treated groups, and persisted in two recovery females. Microscopic abnormalities were detected in the Pituitary where degranulation/hypertrophy of pituicytes, was present in males from all treated groups and in one female treated with 1200 ppm only. This finding had resolved after the recovery period. Follicular epithelial hypertrophy was present in the thyroid glands in animals of either sex from all treated Groups. This change resolved after the recovery period. Abnormalities were shown in the stomach where hyperplasia of the non-glandular (fore) stomach was present in females only treated with 1200 ppm and was reversible after the recovery period. Uterine atrophy was present in some females treated with 1200 and 800 ppm, often associated with anoestrus morphology in the vagina. This was not detected in females after the recovery period. An increase in fat vacuolation in the sternal bone marrow was present in females treated with 1200 and 800 ppm. There was evidence of recovery and although this did not appear to be complete after the treatment free period, the spread in the recovery animals was considered to be within the limits of normal variability.
Dietary concentrations of 500, 800 and 1200 ppm resulted in a clear functional change in animals of either sex at all dose levels. A NOEL has therefore not been established on this study. The adverse systemic effects in the pituitary-thyroid axis were observed at all treatment levels in both sexes, 500 ppm (equivalent to a mean achieved dose of 43.1 mg/kg bw/day for males and 47.4 mg/kg bw/day for females) was considered to be the LOAEL.

Justification for classification or non-classification

The following effects were observed:

- Liver is the organ which show the most effects: From 34/40 mg/kg bw onwards relative weight increase of >=16% along with centrilobular hypertrophy and periportal macrovacuolation (fatty type) (OECD TG 407).

- Thyroid relative weighs are increased at and from 40 mg/kg bw (OECD TG 407) key effect is follicular hypertrophy.

- The effect on the pituitary is puzzling and not straightforward considering the relative and absolute weight: there is an overall decrease starting at 34 mg/kg bw (OECD TG 421 and OECD TG 407) but this is not dose depending. In the OECD TG 407 all treated males show hypertrophy and degranulation while this is seen in one female at 100 mg/kg bw in the OECD TG 407.

- Males sex organs prostate with seminal vesicles show a dose related decrease >=40 mg/kg bw, but not testis. There is a decrease in sperm and sperm motility at 100 mg/kg bw (not significant) in the OECD TG 407.

- Uterus and cervix: there is a dose related decrease and atrophy seen in females in the OECD TG 407 at 67 and 100 mg/kg bw including atrophy and aneustrogenicity (at 40 mg/kg bw this is 12% and without atrophy).

STOT-RE is proposed because the liver effects are indicative for functional changes at 40 mg/kg bw in a subacute study. Whether or not the thyroid, pituitary and reproductive organ effects are primary or secondary and/or reversible, the marked effects are seen at low doses and therefore indicative for STOT-RE.

Based on the available information, classification for specific target organ toxicity upon repeated exposure the substance is classified as STOT RE 2, H373: May cause damage to the organs (liver, thyroid, pituiry, prostate, seminal vesicles, uterus and cervix) through prolonged exposure, in accordance with EU CLP (EC no. 1272/2008 and its amendments)