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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Dose descriptor:
NOAEL
125 mg/kg bw/day
Additional information

Referenced studies:

The following studies are taken into account for the evaluation of the reprotoxic potency because in all cases experimental animals were treated repeatedly by gavage:

- (1) Subacute Oral Toxicity Study with Wistar Rats (according to OECD Guideline No 407)

Contract laboratory: vivo Science GmbH, Fabrikstr. 3, D-48599 Gronau

Study report L09-006, dated 20 May 2009

- (2) Prenatal Developmental Toxicity Study (according to OECD Guideline No 414)

Contract laboratory: vivo Science GmbH, Fabrikstr. 3, D-48599 Gronau

Study report L09-007 dated 04 November 2009

- (3) One Generation Oral Reproduction Toxicity Study in Rats (according to OECD Guideline

No 415),

Contract laboratory: RTC S.p.A., Vito Tito Speri, 12/14; I-00040 Pomezia (Rome)

Study report no. 81940, 2011

Major parameters and findings:

In the following major parameters and findings of the referenced studies are compiled:

Study (1)

- Doses of 0 (vehicle control), 63, 250 and 1,000 mg/kg body mass (bm) per day were administered by oral gavage for 28 days to Wistar rats.

- Minor deviations from control values of some measured parameters in the high dose group could be assigned to be “coincidental” and/or an adaptative type of response.

- Therefore the subacute NOAEL would be 1,000 mg/kg bm/d (see stated opinion of the study director).

- No deviations from control parameters were observed at the low dose group, i.e. 63 mg/kg bm/d, i.e. NOEL 63 mg/kg bm/d.

Study (2)

- Doses of 0 (vehicle control), 10, 50 and 250 mg/kg body mass (bm) per day were administered by oral gavage for days 6 through 20 of gestation to 100 pregnant Wistar rats.

- Marked reduction of body mass was observed in the high dose group for the dams.

- Therefore the contract laboratory stated a maternal NOAEL of 50 mg/kg bm/d.

- For the treated foetuses developmental toxicity/teratogenicity was observed in visceral (eye and blood vessels) and skeletal (ribs) examinations in a dose dependent manner for (nearly) all doses.

- Therefore the testing laboratory stated an embryo-foetal NOAEL of < 10 mg/kg bm/d.

Study (3)

- Doses of 0 (vehicle control), 50, 250 and 1,000 mg/kg body mass (bm) per day for males and of 0 (vehicle control), 1, 5 and 125 mg/kg body mass (bm) per day for females were administered by oral gavage to in total 220 Wistar rats.

Dose levels were chosen based on the outcome of the subacute (1) and the prenatal/de-velopmental toxicity study (2).

- Dosing started 10 weeks for males before mating with females and for females 2 weeks before mating with males; dosing continued for in total 105 days for males until sacrifice and for females during gestation and further for in total 58 days.

- The NOAEL with respect to reproduction toxicity for males was stated by the study director to be 1,000 mg/kg bm/d and for females 125 mg/kg bm/d.

- The NOAEL with respect to developmental toxicity is higher than the highest tested dose of 125 mg/kg bm/d.

- Any developmental toxicity/teratogenicity in visceral (eye and blood vessels) and skeletal (ribs) examinations of the foetuses was not detected.

Discussion of major and (partly) contradictory results of the studies:

Preliminarily it should be emphasized that it is a priori assumed that with respect to proper dosing no errors occurred during preparation of the test substance formulation to be applied to the experimental animals by gavage.

It is noted that while no analytical measurements were conducted for the subacute and prenatal/developmental toxicity studies, an analytical verification of the test substance formulations was performed for the one generation study!

1. Significant toxicity to males and females with regard to reproductive organs was not observed in the subacute (1) and one generation study (3) up to the highest dose of 1,000 mg/kg/d (both for males and females) and 125 mg/kg/d (for females) respectively, whereas maternal toxicity seems to be higher by comparison of the stated NOAELs in the prenatal/developmental toxicity (2) than in the one generation study (3).

This means that while the degree of toxicity derived from the subacute study (1) is very much comparable to the degree of toxicity in the one generation study (3) (only an influence on body weight was observed in males with the longer duration of the one generation study), a marked reduction of the respective maternal NOAELs was observed in the prenatal/developmental toxicity (2) (i.e. 50 mg/kg/d) versus the one generation study (3) (i.e. > 125 mg/kg/d).

2. The prenatal/developmental toxicity study (2) revealed a treatment related decrease of the

body mass gain of non-gravid female rats at the end of the in-life phase and an increase (!) of the body mass gain of pregnant rats in the low and mid dose group, but a decrease in the high dose group, i.e. no plausible dose dependency during the treatment period is obviously observed.

3. The prenatal/developmental toxicity study (2) noted a post-implantation loss of 2.5 % in the low dose (10 mg/kg/d), 3.1 % in the mid-dose (50 mg/kg/d) and 6.3 % in the high dose group (250 mg/kg/d) compared with 1.3 % in the control group which again does not indicate a clear and plausible dose dependency of these adverse effects.

4. Foetus and placenta masses were reduced in the prenatal/developmental toxicity study (2) significantly at 50 and 250 mg/kg/d which was not the case in the one generation study (3) up to the highest dose of 250 mg/kg/d.

Again the toxicity profile derived from the prenatal/developmental toxicity (2) does not fit to the results of the one generation study (3).

5. The prenatal/developmental toxicity study (2) detected severe (!) treatment related effects on foetuses at already the lowest dose of 10 mg/kg/d (citation from study report):

“Malformations of the eye occurred in foetuses of all three dose groups. The malformations included retina folds, lens alteration, aphakia, and anophthalmia. The incidences increased with increasing dose. None of the malformations occurred in the control group (0 mg/kg/d). Malformations of the great blood vessels occurred in one foetus of the control (0 mg/kg/d), three foetuses of the medium dose (50 mg/kg/d) and five foetuses of the high dose group (250 mg/kg/d). The malformations included absent innominate artery (in one foetus of the control, two foetuses of the medium dose, and three foetuses of the high dose group) as well as right-sided aortic arch (in one foetus of the medium dose and two foetuses of the high dose group). …

The incidence of incompletely ossified temporal, frontal, supraoccipital, squamosal, hyoid, and zygomatic bones was increased in the high dose group (250 mg/kg/d). Dam no. 750 of the high dose group (250 mg/kg/d) had six late resorptions and six living foetuses with an extremely low foetal body weight resulting in incomplete ossifications of several bones. All foetuses had not ossified metacarpal and metatarsal bones. Foetus no. 750/2 (foetal body weight of 2.90 g) had misshapen scapulas, humeri, radii, and ulnas, as well as misshapen fibulas and tibias. Foetus no. 750/4 (foetal body weight of 3.35 g) had misshapen scapulas, humeri, radii, and ulnas, as well as misshapen right femur. Foetus no. 750/9 (foetal body weight of 2.87 g) had misshapen scapulas, humeri, radii, and ulnas. The incidence of incompletely ossified interparietal bones was increased in all three dose groups without a clear dose-dependency, while the incidence of incompletely ossified parietal bones as well as of thickened and wavy ribs was dose-dependently increased in all three dose groups.”

whereas the one generation study (3) did not detect any (!) treatment related (developmental/teratogenic) effects on the foetuses up to the highest dose of 125 mg/kg/d.

Again the toxicity profile according to the prenatal/developmental toxicity (2) with respect to embryo-foetal adverse effects is extremely divers from the results of the one generation study (3).

6. The prenatal/developmental toxicity study (2) noted a possibly (?) relevant deviation from the study plan (citation):

„The foetuses had to be re-fixated at the Principal Investigator for at least 5 days using Davidson solution containing 10% formalin as originally stated in SOP RDP/R2 07.03 of the PI, since fixation of Wilson foetuses at the test facility was performed using a formalin content of 4% instead of 10% by error. Therefore, the start of foetal evaluation of the remaining Wilson foetuses had to be postponed

from CW 16 to CW 18 2009. In order to minimise the risk of total data loss due to possible staining artefacts, the staining of Dawson foetuses was performed subsequently in cohorts of 10-30 litters. As a result, shipment of foetuses and the start of evaluation at the PI were delayed as stated above. This change did not lead to any alterations concerning the content, or results of study L09-007“

Note, that the underline is from the authors!

It remains unclear if such deviation might have caused artefacts during the staining procedure.

7. The prenatal/developmental toxicity study (2) noted a further possibly (?) relevant deviation from the study plan because the animals of the low and the mid dose group arrived about 14 days later at the testing site than the animals of the vehicle and the high dose group, i.e. testing did not start at the same day!

Also it is noted that the mating of the experimental animals occurred at the supplier`s facilities and already pregnant animals were received at the testing facility.

It remains unclear whether such deviations might have had an influence on the study integrity, but these shortcomings shed doubts on the study results.

8. Whereas in the one generation study (3) the health of the experimental animals was checked by a full post mortem examination of 5 animals (all animals were stated to be healthy at the beginning of the study) no such health check was performed prior to the execution of the prenatal/developmental toxicity study (3).

9. The water consumption of the animals in the prenatal/developmental toxicity study (2) was remarkably high: Up to 23 % above control for the high dose group, but also in the other dose groups the water consumption increased in a dose depended manner.

Again this observation was neither made in the subacute (1) nor in the one generation study (3), therefore the respective toxicity profiles do not “fit”.

Conclusions:

The substance BHT seems to be devoid of any reproductive toxicity to male and female Wistar rats with regard to reproductive organs and mating behaviour according to the subacute (1) and the one generation study (3).

Maternal effects in pregnant Wistar rats dosed during gestation were only observed in the prenatal/developmental toxicity (2) but not in the one generation study (3) the fact of which cannot be plausibly explained.

Embryo-foetal adverse effects, i.e. developmental/teratogenic toxicity, in the offspring of treated dams during gestation occurred only in the prenatal/developmental toxicity study (2), though distinctly below the maternal toxicity threshold.

Although these effects were severe already in the low dose group of the prenatal/developmental toxicity study (2) they were not observed at much higher dose levels in the one generation study (3). Also minor toxic effects, which were reported in the prenatal/developmental toxicity study (2) were not reported in the one generation study (3).

Overall the observed toxicity profiles from the subacute (1) and the one generation study (3) do not “fit” to the study results of the prenatal/developmental toxicity study (2).

Since certain shortcomings are to be noted with the execution of the prenatal/developmental toxicity study (2) as described above in more detail:

• No verification of nominal dose levels by analytical measurements

• No dose-effect dependency for various test parameters

• Critically (?) flawed staining procedure of foetuses

• Non-coincidental start of investigation and mating outside the contracted test facility

• No health check of received experimental animals

and since the execution of the one generation study (3) was devoid of any such shortcomings at all and since its results do plausibly fit to the findings of the subacute study (1) it is concluded, that the biological findings of the prenatal/developmental toxicity study (2) may be of an artefactual nature; overall it is concluded that the results from the one generation study (3) seem to be of much higher validity and relevance than from the prenatal/developmental toxicity study (2).

It should also be considered, that the application of the substance in the one generation study (3) was longer and the number of treated animals much higher than in the prenatal/developmental toxicity study (2).

It is therefore finally concluded, that the observed maternal and developmental/teratogenic effects as observed from the prenatal/developmental toxicity study (2) are of very doubtful relevance for humans.

The relevant NOAELs after oral uptake of BHT for reproduction for any risk assessment should be derived from the one generation study (3) and set to 1,000 mg/kg/d for male rats, 125 mg/kg/d for female rats and 125 mg/kg/d for the offspring.

Justification for classification or non-classification