Indeno[4,3a-b]furan, decahydro-2,2,7,7,8,9,9-heptamethyl-

Endpoint summary

Description of key information

Oral: NOAEL = 150 mg/kg bw/day, rats (OECD TG 407 and OECD TG 421, GLP).

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

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two GLP compliant oral studies (OECD TG 407 and 421) were available, both assigned a Klimisch 1 rating, adequately covering this endpoint.

Additional information

OECD TG 407

A 28-day repeated dose toxicity study according to OECD TG 407 and under GLP conditions was performed to determine the effect of Amber Xtreme on rats. Doses of 1000, 150 and 15 mg/kg bw/day were administered by oral gavage. A satellite high dose group was allowed to recover for 14 days after treatment. The following observations were made during the treatment and recovery period: clinical signs, body weight, food and water consumption. Upon termination several haematology, clinical chemistry and urine parameters were checked for all animals. At necropsy, organs were weighed and prepared for histopathology.

Results: No mortality was observed throughout the study. Episodes of increased salivation around the time of dosing were detected primarily in the high dose group, as well as some incidental findings like red/brown staining of the fur around the eyes. A statistically significant reduction in bodyweight gain was observed in the high dose group for females. In absence of effects on body weight this was not considered adverse. Furthermore, a statistically significant increase in prothrombin time was detected in males treated with 1000 mg/kg/day (+68%). A statistically significant increase in plasma cholesterol levels was observed in males and females treated with 1000 mg/kg/day: -31% and +60%, respectively. In females a 2.8x increase in gamma glutamyl transpeptidase was seen in the high dose. (Absolute and) relative liver weight increase statistically significant were observed for male and female animals of the high dose group (31 and 41%, respectively) and in females of the mid-dose group (18.5%). Slight centrilobular hepatocytes hypertrophy was seen in males at 1000 mg/kg bw (5/5) and in females in the mid and high dose group (3/5 and 5/5, respectively). Recovery was apparent after 14 days. Slight follicular cell hypertrophy was observed in the thyroid glands of both sexes of the high dose group 4/5 males and 5/5 females. No other treatment-related effects were noted.

The results of this study show that 28-day gavage treatment of rats with 1000 mg/kg bw/day Amber Xtreme showed increased prothrombine time in males at the high dose. There were alteration of clinical chemistry for cholesterol in both sexes and gamma GT in females. These effects were associated with increased relative liver weights accompanied with centrilobular liver hypertrophy at 1000 mg/kg bw. At the mid dose level of 150 mg/kg bw/day similar effects were noted but to a lesser extent. The No Observed Adverse Effect Level (NOAEL) was set at 15 mg/kg bw/day based on dose related finding in the liver resulting in increased liver weights starting in the mid dose with accompanying increased liver parameters and centrilobular hypertrophy.

Dose Range Finder OECD TG 421

For the Reproscreen study a 14-day dose range finder was performed at 225, 1125, 2250 and 6750 mg/kg diet which are nominal 15, 75, 150 and 450 mg/kg bw. The dietary route was selected to prevent effects due to bolus dosing. At 2250 and 6750 ppm, increased relative liver weights were seen in males +26 and 38% and females +19 and +41%, respectively. These adverse relative liver weights were used to set a maximum dose level of 1500 ppm to prevent repeated dose effects that could possibly obscure reproductive effects.

OECD TG 421

The supporting study (for repeated dose toxicity) was a reproduction/developmental toxicity screening test that was performed according to OECD TG 421 and under GLP conditions. Twelve Wistar rats/sex/dose received diets containing Amber Xtreme at concentrations of 0, 225, 750 or 1500 mg Amber Xtreme/kg diet (equivalent to 15, 50, and 100 mg/kg bw/day nominal) during a premating period of 2 weeks and during mating (1 week), gestation and lactation until postnatal day 4. Amber Xtreme was homogeneously distributed in the diets and stable in diet stored in an open container for 24 hours, except for the high-dose level (-11%). When stored in a closed container in the freezer (< -18°C), the high dose of Amber Xtreme in diet was stable for 5 weeks. The concentration of Amber Xtreme was lower than intended in all diets. Relative differences of -28 %, -26 % and -14 % were observed for the measured concentrations in the low-, the mid- and the high dose group, respectively. As a result, the maximum intake of the highest dose group was 64 and 83 mg/kg bw, for males and females, respectively.

Results: Daily clinical observations did not reveal any treatment-related changes in the animal's appearance, general condition or behaviour. No treatment-related effects were observed in mean body weight, body weight changes and food consumption throughout the study. Relative liver weight was statistically significant increased with 10.6 and 12.7% in mid dose and high dose males, respectively, but not in females. The toxicological relevance of this finding is doubtful, because there was no clear dose-response and no histopathological changes in the liver were observed. Macroscopic and microscopic examination of organs did not reveal any treatment-related changes. For males and females the NOAEL for systemic toxicity is established at 1500 mg Amber Xtreme/kg diet (corresponding to a dose of at least 64 and 83 mg/kg bw/day, respectively), because no clear effects on systemic toxicity were observed.

Conclusion

The results of the OECD TG 407 show that relative liver weights at 150 mg/kg bw were 12 and 18% increased in males and females, respectively, which was statistically significant in females. As this increase in relative liver weight is below 20%, it is considered to be an adaptive response. However, at this dose level, minimal centrilobular hepatocyte enlargement was observed in all male animals and in 3 out of 5 female animals. Therefore in this test the NOAEL was set at 15 mg/kg bw/day. In the OECD TG 421, relative liver weight was statistically significant increased with 10.6 and 12.7% in mid dose and high dose males, respectively. As there were no histopathological changes observed, these relative liver weight increases were not considered to be adverse and the NOAEL was set to 64 mg/kg bw the highest dose tested. Based on the OECD 407, it can be concluded that the actual NOAEL would be somewhere in between 15 and 150 mg/kg bw. As in the OECD TG 421 the NOAEL is at least 64 mg/kg bw, this NOAEL was used in the DNEL derivation.

Justification for classification or non-classification

The test substance does not have to be classified for repeated dose toxicity according to EU CLP (EC No. 1272/2008 and its amendments).