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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Remarks:
In vivo mechanistic data for endocrine activity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Experimental information for endocrine activity discussion.

Data source

Reference
Reference Type:
publication
Title:
. Comparison of effects of estradiol (E2) with those of octylmethoxycinnamate and 4-methylbenzylidene camphor on fat tissue, lipids and pituitary hormones.
Author:
Seidlova-Wuttke D, Christoffel J, Rimoldi G, Jarry H, Wuttke W.
Year:
2006
Bibliographic source:
Toxicology and Applied Pharmacology 214: 1-7

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A study in ovariectomised (ovx) Sprague Dawley rats compareing the effects of estradiol with those of 4-methylbenzylidene camphor on fat tissue, uterine weight, lipids, and pituitary hormones
GLP compliance:
not specified
Remarks:
Published study from a peer-reviewed journal.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Obtained from Sigma, Diesenhofen, Germany

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
- Ovariectomised (ovx ) Sprague Dawley rats (250g +/- 5g)
- Maintained on pelleted chow in which soy proteins replaced by potato proteins
- Lighting from 06.00 to 18.00, a room temperature of 25oC with 55% humidity

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
low dose: 59.27 mg/d (approx 237 mg/kg bw/d); high dose:285.37 mg/d (approx 1142 mg/kg bw/d);
E2: 0.445 mg/d (approximately 1.8 mg/kg bw/d) as positive control
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
12 weeks
Frequency of treatment:
Daily
Duration of test:
12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
237 mg/kg bw/day (nominal)
Remarks:
Nominal concentration adjusted based on food intake
Dose / conc.:
1 142 mg/kg bw/day (nominal)
Remarks:
Nominal concentration adjusted based on food intake
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment
other: Positive control, concurrent
Details on study design:
Sprague Dawley rats rats (250g +/- 5g) in groups of 12 were administered non-soy containing feed with added 4-methylbenzylidene camphor to provide nominal doses of 50 mg/day and 250 mg/day for 12 weeks. E2 was also tested as a positive control plus an untreated control group. Body weight and vaginal smear taken twice weekly. Animals were ovariectomised before treatment.
Paratibial fat depot in hind leg (considered by the authors to be extremely sensitive to withdrawal of oestrogens) was measured by computerised tomography (CT). Blood was taken for serum analysis for lutenising hormone (LH), thyroid stimulating hormone (TSH), T3 and T4), and Leptin and estradiol which were measured by radioimmuno assay (RIA). Serum cholesterol, HDL, LDL and triglycerides were assayed by enzymatic analyses.
Statistics:
One way ANOVA and Dunnett’s post hoc test for multiple comparisons were used as statistical tests with p <0.05 considered to be significant

Results and discussion

Observed effects

The low dose of 4-methylbenzylidene camphor had a small but significant effect on uterine weight, the high dose was not significantly different from the control, whilst E2 had a large effect on uterine weight increase. Body weight gain was also significantly decreased by E2 and 4-methylbenzylidene camphor, although the effect of E2 was much greater. A significant decrease in fat depot was observed, consistent with the E2 treatment but only at the low 4-methylbenzylidene camphor dose. Serum leptin levels were significantly reduced at both doses of 4-methylbenzylidene camphor and with E2.
The mean E2 serum level in E2 treated rats was 84 ± 58 pg/ml. E2 levels were not detectable in 4-methylbenzylidene camphor treated rats or in negative controls. Serum LH was significantly increased by 4-methylbenzylidene camphor at both doses. In contrast, serum LH levels were decreased by E2, which is consistent with the known negative feedback effect of E2 on this hormone.
Exposure to E2 significantly reduced serum cholesterol, HDL and LDL levels, but were not effected by 4-methylbenzylidene camphor except for a significant increase in serum HDL levels at the low dose. Neither E2 or 4-methylbenzylidene camphor had a significant effect on serum triglycerides.
Treatment with E2 did not have any effects on TSH, T3 or T4 level, whereas treatment with 4-methylbenzylidene camphor significantly increased TSH levels, and reduced T4 levels at both doses.

Applicant's summary and conclusion

Conclusions:
Weak oestrogenic activity of 4-methylbenzylidene camphor was demonstrated only at the low dose (statistically significantly) by small effects on uterine weight, body weight, and serum levels of leptin. In a parallel in vitro study 4-methylbenzylidene camphor did bind to ERβ and the cytosolic preparation and therefore receptor mediated activity cannot be ruled out. However, this and other effects observed on the endocrine system (LH stimulation as opposed to inhibition seen with E2 and effects of thyroid hormones) may be via an alternative mechanism of action based on neurotransmitters.
Executive summary:

A study was conducted in ovariectomised (ovx) Sprague Dawley rats to compare the effects of estradiol with those of 4-methylbenzylidene camphor on fat tissue, uterine weight, lipids, and pituitary hormones. Groups of 12 rats were administered non-soy containing feed with added of 4-methylbenzylidene camphor to provide nominal doses of 50 mg/day and 250 mg/day for 12 weeks. E2 was also tested as a positive control and an untreated control group was included. Animals were ovariectomised before treatment. Body weight and vaginal smears were taken twice weekly. Exposures calculated from feed intake and the amount of test substance added to feed were used to calculate average intakes which were approximately 237 mg/kg bw/day for the low 4-methylbenzylidene camphor dose (59.27 mg/day), approximately 1142 mg/kg bw/day for the high 4-methylbenzylidene camphor dose (285.37 mg/day), and approximately 1.8 mg/kg bw/day for the positive E2 control (0.445 mg/day). The authors also investigated the ERαand ERβbinding affinity of 4-methylbenzylidene camphor in vitro. These in vitro results are reported in IUCLID 7.12.   

Paratibial fat depot in the hind leg, considered by the authors to be extremely sensitive to withdrawal of estrogens, was measured by computerised tomography (CT). Blood was taken for serum analysis for luteinising hormone (LH), thyroid stimulating hormone (TSH), T3, T4, and E2, which were measured by radioimmunoassay (RIA). Serum cholesterol, HDL, LDL and triglycerides were assayed by enzymatic analyses.

The low dose of 4-methylbenzylidene camphor had a significant but small effect on uterine weight; the high dose was not significantly different from the control, whilst E2 had a large effect on uterine weight increase. Body weight gain was significantly decreased by E2 (effect of E2 was much greater) and 4-methylbenzylidene camphor. A significant decrease in fat depot was observed, consistent with the E2 treatment but only at the low 4-methylbenzylidene camphor dose. Serum leptin levels were significantly reduced at both doses of 4-methylbenzylidene camphor and with E2.

The mean E2 serum level in E2 treated rats was 84 ± 58 pg/ml. E2 levels were not detectable in 4-methylbenzylidene camphor treated rats or in negative controls. Serum LH was significantly increased by 4-methylbenzylidene camphor at both doses. In contrast, serum LH levels were decreased by E2, which is consistent with the known negative feedback effect of E2 on this hormone.

Exposure to E2 significantly reduced serum cholesterol, HDL and LDL levels, but were not effected by 4-methylbenzylidene camphor except for a significant increase in serum HDL levels at the low dose. Neither E2 nor 4-methylbenzylidene camphor had a significant effect on serum triglycerides.

Treatment with E2 did not have any effects on TSH, T3 or T4 level, whereas treatment with 4-methylbenzylidene camphor significantly increased TSH levels, and reduced T4 levels at both doses. The authors speculate that the stimulated TSH levels may be due to activation of thyroid deiodinase by 4-methylbenzylidene camphor explaining increased (albeit not significantly) serum concentrations of T3. 

In summary, a weak estrogenic activity of 4-methylbenzylidene camphor was demonstrated by small effect on uterine weight (only significantly at the low dose), body weight, and serum levels of leptin. In a parallel in vitro study 4-methylbenzylidene camphor did bind to ERβand the cytosolic preparation and therefore receptor mediated activity cannot be ruled out. However, this and other effects observed on the endocrine system (LH stimulation as opposed to inhibition seen with E2) may be via a mechanism not involving binding to estrogen receptors. The authors suggest that the action of 4-methylbenzylidene camphor on lipid metabolism and thyroid hormones could be due to neurotransmitter-involving mechanisms and needs to be further explored.

The study provides some evidence of weak estrogen-like activity, but it is unlikely that the effects of 4-methylbenzylidene camphor are mediated through similar mechanisms to E2.

The study is allocated a reliability rating of 2: reliable with restrictions, as the non-standard in vivo study protocol is provided in sufficient detail to accept the data, although only two dose levels were tested.Thestudyfalls in Level 3 of the OECD CF.