Registration Dossier

Administrative data

Description of key information

In the chosen key study according to OECD TG 408 and GLP, substance-related effects were observed at dose levels of 4500 ppm and

15000 ppm, corresponding to 340 and 1085 mg/kg bw/day. Identified target organs were the liver, thyroid and pituitary gland as a secondary consequence of hepatic enzyme induction. The slight changes seen in the red blood cells of the high dose females are probably related to a marginal adverse effect of the test substance at high dose level. Based upon the above mentioned findings, the no observed adverse effect level under the conditions of this study is set at 175 mg/kg bw/day for males and females.

Key value for chemical safety assessment

Additional information

In the chosen key study according to OECD TG 408 and GLP, octocrilene was administered at doses of 750, 2250, 4500 and 15000 ppm to 10 Wistar rats per sex and dose for 3 months in the diet, corresponding to approx. 58, 175, 340, 1085 mg/kg bw/day ingested test substance, respectively (BASF 50S0227/92059).

Animals receiving 15000 ppm octocrilene showed a reduced food consumption (13% in males and 10% in females), reduced body weight/ body weight gain (10%/16% in males and 8%/15% in females). Urine specimens showed a yellow discoloration in both sexes. Alkaline phosphatase, total protein, globulins and total cholesterol was increased in females and gamma-glutamyltransferase was increased in both sexes. Total bilirubin was decreased in both sexes and a female specific decrease of alanine aminotransferase and aspartate aminotransferase was observed. Increased absolute and relative liver weights along with prominent acinar pattern and hypertrophy of periacinar and centriacinar hepatocytes in both sexes were observed. Increase in platelets in both sexes, shortened prothrombin time in females and a decrease in hemoglobin mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration in females was observed. The thyroid follicular epithelium in both sexes showed slight to moderate hypertrophy associated with pale staining colloid and an increased number of hypertrophic cells in the pituitary gland of males was observed.

In the 4500 ppm group, a decrease in total bilirubin (both sexes) and a decrease in alanine aminotransferase and aspartate aminotransferase was observed in females. Absolute and relative liver weights were increased in females only, however a hypertrophy of periacinar and centriacinar hepatocytes was also observed in males. In females, platelets, total protein and globulins were found to be increased. Minimal or slight hypertrophy of the thyroid follicular epithelium and associated pale staining colloid in both sexes was observed in this dose group. In the other dose groups, i.e. 2250 ppm and 750 ppm, no substance-related findings were recorded.

Taken together, substance-related effects were observed at dose levels of 4500 ppm and 15000 ppm, corresponding to 340 and 1085 mg/kg bw/day. Identified target organs were the liver, thyroid and pituitary gland as a secondary consequence of hepatic enzyme induction. The slight changes seen in the red blood cells of the high dose females are probably related to a marginal adverse effect of the test substance at high dose level. Based upon the findings mentioned above, the no observed adverse effect level under the conditions of this study is set at 175 mg/kg bw/day for males and females.

In a supporting mechanistic subacute toxicity study in rats similar to OECD TG 407 and according to GLP, the thyroid effects of octocrilene via enzyme induction in the liver was assessed (BASF 2019; 99C0495/00S048). Octocrilene was administered via diet to groups of 5 Wistar rats per sex and dose at concentrations of 0, 1000 ppm, 3000 ppm and 10000 ppm over a period of 14 days (Subset B) and 28 days (Subset A), corresponding to 63-72, 188-215 and 630-720 mg/kg bw/day ingested octocrilene, respectively. During the course of the study, blood samples were taken and examined for thyroid hormone levels (T3, T4 and TSH; Subsets A and B). Blood samples for the analyzes of hormone levels (T3, T4, TSH) were taken on study days -3, 7, 21 and 29 for all animals of Subset A and on study days -3, 0, 3 and 14 for all animals of Subset B. Shortly before necropsy, blood samples were taken for the examination of hematology and clinical chemistry parameters (Subset A, only). At necropsy, organ weights were determined, and histopathological examinations of selected organs were performed (Subsets A and B). Parts of the liver tissue were fixed for histopathology, others were deep frozen in liquid nitrogen for bioanalytical examinations. For bioanalytical examinations (Subset B), liver microsomes were prepared and characterized for their total cytochrome P450-content as well as for the activities of Ethoxyresorufin-O-deethylase (EROD), Pentoxyresorufin-O-depentylase (PROD), Benzyloxyresorufin-O-debenzylase (BROD), 4-Methylumbeliferone glucuronosyltransferase (MUF-GT), 4-Hydroxybiphenyl-glucuronosyltransferase (HOBI-GT) and the T4-specific UDP-glucuronosyltransferase. In addition, the activity of Thyroxin 5’-deiodinase (Iodothyronine deiodinase) type D1 and D3 was assessed in an external laboratory without GLP status.

Lower mean body weights and body weight change values occurred in high dose group male and female animals (treatment for 28 days) as well as in male animals (treatment for 14 days). These changes were assessed to be related to treatment and considered as adverse. No clinical signs of systemic toxicity were observed in high dose group female animals (treatment for 14 days) as well as low and mid dose group male and female animals.

In high dose group females, increased γ-glutamyl transferase (GGT) activities as well as higher urea, cholesterol and triglyceride levels indicated a changed liver cell metabolism. Higher urea values were also observed in high dose group males which was most probably due to an increased protein metabolism in the liver cells.

Higher serum TSH levels in high dose rats of both sexes on study days 21 and 29 as well as in high dose females already on study day 14 were observed, whereas no significant changes occurred for measured T3- and T4-levels in any test group or timepoint assessed.

The increase in TSH levels in combination with physiological T4 hormone levels were due to a compensation of the increased liver-related thyroid hormone clearance. This resulted in an increased synthesis of the hormones induced by a stimulation of the thyroid via the pituitary gland.

After a treatment period of 14 days, high dose group animals showed absolute and relative weight increases of the liver in males (absolute / relative: +12.85% / +23.05%) and females (absolute / relative: +21.88% / +27.20%). These changes were assumed to be treatment-related and potentially adverse. They were consistent with minimal diffuse hepatocellular hypertrophy (in 1 / 5 males, and 3 / 5 females) and with a microsomal liver enzyme induction. Furthermore, the thyroid glands of high dose group males (2 / 5) and females (3 / 5) revealed a minimal follicular cell hypertrophy/hyperplasia, accompanied by minimal altered colloid. In females, an association with the increased levels of TSH and the minimal histopathological changes in the thyroid is more evident than in male animals. In the mid dose group, a minimal increase of the relative liver weight of females was noted. However, since no histopathological correlate was noted, this change was regarded as possibly treatment-related and not adverse.

After a treatment period of 28 days, the high dose group showed a decrease of the final body weight in males (-10.8%) and an increase of the relative liver weights of males (+25.63%) and females (+23.69%), which were regarded as treatment-related and consistent with a microsomal enzyme induction. No histopathological correlate and no adverse clinical chemical liver parameters were noted in males. Therefore, the relative weight increase in males was assumed to be treatment-related but not adverse. In females, a minimal hepatocellular diffuse hepatocellular hypertrophy (2 / 5 females) correlated with the relative liver weight increase. Since these changes were accompanied by altered clinical chemical parameters, they were considered treatment-related and adverse. In addition, the thyroid glands of high dose group males (2 / 5) and females (3 / 5) revealed a minimal follicular cell hypertrophy/hyperplasia, accompanied by altered colloid (minimal to slight in males and minimal in females). These changes were most likely associated with the increased TSH levels measured in males and females.

Total CYP 450 contents in liver microsomes of high dose group male Wistar rats were significantly increased when compared to untreated controls. In parallel to this finding, CYP 1A-related EROD activities were increased significantly in high dose group male rats. However, an induction factor below 2 is assessed to be low for EROD-activity. There was no effect of octocrilene treatment on total CYP 450 contents or EROD-activities in female rats. In mid and high dose group male and female rats, CYP 2B-related PROD-activities were significantly higher when compared to untreated animals. The induction of PROD-activity by octocrilene treatment is up to about 10- and 6-fold in males and females, respectively and is assessed to be biologically relevant. In parallel to this finding, also BROD-activities were significantly and dose-dependently increased for mid and high dose male and female rats when compared to untreated animals. For male animals, maximum increase in BROD activity was about 6-fold. For female animals, BROD-induction was even more pronounced when compared to untreated controls for which BROD-activity was below the limit of quantification. Effects on glucuronosyltransferase activities consisted of significantly increased MUF-GT and HOBI-GT-activities in high dose group male and female rats when compared to untreated controls and the maximum induction was about 2-fold. T4-specific UDP-glucuronosyltransferase activity was significantly increased for mid and high dose females by induction factors of 1.5 and 3.1, respectively. For high dose males, T4-GT-activity was significantly increased (1.9-fold) when compared to untreated controls.

Thyroxin 5’-deiodinase type 1 activity was significantly reduced in high dose males (-32% compared to controls) whereas Thyroxin 5’-deiodinase type 3 activity was significantly induced (2 fold compared to controls) in mid and high dose male animals.  However, based on measured levels of T3 and T4 that were not altered at any sampling time point within the current study, the changes of activities of Thyroxin 5’-deiodinase type 1 and Thyroxin 5’-deiodinase type 3 were assessed as non-adverse.

It has been established in literature, that hepatic microsomal enzyme inducers disrupt T4 homeostasis by increasing T4 glucuronidation, which is an important metabolic pathway for T4 in rats (Hood et al. 2000). For example, phenobarbital has been shown to increase UDP-GT activity toward T4 and to increase T4 biliary excretion. Accordingly, other microsomal inducers (pregnenolone-16alpha-carbonitril (PCN), 3-Methylchloranthrene, Aroclor 1254) result in reduced T4 levels based on the induction of T4 UDP-GT activity, with minimal effect on T3 levels. Furthermore, Phenobarbital and PCN increase serum TSH levels. Liver Diodinase 1 activity was reduced by these substances (up to 75%) and the authors propose, that the reduced ORD activity in the liver is a physiological response to the hypothyroid state (i.e. lowered serum T4 levels) of the treated animals.

In the mechanistic study with octocrilene, an increase in TSH levels but no significant effects on T3 and T4 have been found. No such increases in TSH levels and no significant changes in T4 levels were observed in the recently performed EOGRTS with octocrilene (BASF 2019; 03R0495/00X056). Compared to the effects on liver deiodinase type 1 after treatment with the other hepatic microsomal enzyme inducers cited, the effect of octocrilene treatment (reduction by 20-30%) was only observed in one sex and rather mild, since a reduction in T4 levels was not evident. Furthermore, the relevance of the 2-fold increase of deiodinase type 3 activity in male livers only remains obscure, given that T3 and T4 levels were not altered, and could represent a further physiological response to altered TSH levels due to the hepatic enzyme induction.

In conclusion, the mechanistic study in Wistar rats with oral administration of octocrilene up to 10000 ppm showed an induction of liver enzymes (PROD, BROD, T4-specific UDP-glucuronosyltransferase) accelerating the thyroid hormone clearance in both sexes. As a result, a compensating positive feedback mechanism was installed, leading to higher TSH levels and hypertrophy/hyperplasia of follicular thyroid gland cells. Accordingly, T3 and T4 levels remained in a physiological range when octocrilene was administered nearly at limit dose (10000 ppm).

In a dermal subchronic study, reported as summary in a publication, 5 male and 5 female New Zealand white rabbits per group were treated topically (open) with octocrilene (130, 264, 534 mg/kg/day) in a mixture of petrolatum and C12-15 alkylbenzoate for 13 weeks (5 days per week, 65 applications in total; Odio 1994). Slight to moderate dose-dependent skin irritation (predominantly erythema and desquamation) at the site of compound application was observed for all dose groups, correlated with a mild depression in body weight gain. However, no evidence for hematological or macroscopic and histopathological abnormalities were observed in all internal organs. Specific investigation of male reproductive functions, i.e. histopathological evaluations of testicles and epididymis and sperm analysis, did not give evidence for treatment related adverse effects.

 

References

Hood A., Klaassen C.D. (2000). Effects of Microsomal Enzyme Inducers on Outer-Ring Deiodinase Activity toward Thyroid Hormones in Various Rat Tissues. Toxicology and Applied Pharmacology 163, 240–248

Justification for classification or non-classification

The present data on repeated dose toxicity do not fulfill the criteria laid down in 67/548/EEC and CLP, and therefore, a non-classification is warranted.