Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction_Main study (EOGRTS OECD 443)_ (The study was requested by the European Chemicals Agency (ECHA) decision number TPE-D-2114407364-56-01/F).

The influence of D,L-Menthol on reproductive performance was assessed when administered continuously in the diet to CD rats. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, an evaluation of the maturing reproductive tract and its integrity and function was performed.

In the F0 generation, three groups of 25 male rats received D,L-Menthol orally, via the diet, at concentrations of 4000, 8000 or 16000 ppm for ten weeks before pairing and until termination. In addition, three groups of 25 female rats received D,L-Menthol orally, via the diet, at concentrations of 4000, 8000 or 16000 ppm for ten weeks before pairing, throughout pairing and gestation, and at concentrations of 2000, 4000 or 8000 ppm during lactation. Corn oil was added to the premix as a stabilizer at a ratio of 5:1 test item:corn oil. A similarly constituted Control group received untreated basal diet with added corn oil for the same duration.

In theF1generation, 40 males and 40 females were treated from weaning to their scheduled termination (relevant to each cohort). From weaning to Week 4 of the formal F1 generation the dietary concentrations were 2000, 4000 and 8000 ppm. For Weeks 5 and 6 of the formal F1 generation the dietary concentrations were 2500, 5000 and 10000 ppm. From Week 7 of the formal F1 generation until termination the dietary concentrations were 4000, 8000 and 16000 ppm, with the exception of the

lactation period for the Cohort 1B females when the dietary concentrations were 2000, 4000 and 8000 ppm. A similarly constituted Control group received untreated basal diet with added corn oil for the same duration.

The dietary concentrations employed were selected in an attempt to achieve an equivalent high dose level of 1000 mg/kg bw/day. Diet concentrations were reduced during lactation to compensate for lactation-specific increases in food consumption. Similarly, during the early stages of the formal F1 generation, the dietary concentrations were steadily increased from lactation levels to adult levels in order to avoid excessively high achieved dose levels.

Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis, urinalysis and immunophenotyping in spleen lymphocytes), growth, development and function of the offspring.

At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation and reproductive organ integrity and function.

The cohorts were as follows:

Cohort

 

 Designation

Animals/Cohort 

Sexual maturation

assessment

Approximate age at

necropsy

 1A

Reproductive &

general toxicity

 20M + 20F

 Yes

 13 weeks

 1B

Reproductive

toxicity

 20M + 20F

 Yes

 23 weeks

Results:

There was no effect of D,L-Menthol administration at any dietary inclusion level investigated on serum T4 or TSH concentrations in F0 or F1 Cohort 1A adult animals, or in the F1 offspring on Day 22 of age.

D,L-Menthol administration at dietary inclusion levels up to and including 16000 ppm was well tolerated by F0 and F1 animals throughout the study, with no test item-related premature deaths or test item‑related changes in general clinical condition, and estrous cycle regularity of the F0 and the F1 Cohort 1A females, and pre-coital interval, mating performance, conception rate and fertility index of the F0 and F1 Cohort 1B animals were unaffected. Similarly, there was no effect of treatment on the

duration between vaginal opening and first estrus in the F1 Cohort 1A females or the stage of estrus at termination of the F0 or the F1 Cohort 1A and 1B females, haematological parameters in F0 and F1 Cohort 1A animals or sperm motility/counts/morphology of the F0 and F1 Cohort 1A males. Macroscopic examination of the F0 animals, the F1 Cohort 1A females and F1 Cohort 1B animals at scheduled termination did not reveal any test item-related abnormalities. There were also no observable effects on the immunophenotyping parameters measured in spleen leukocytes of the F1 Cohort 1A animals.

Kidney weights were increased at the end of the treatment period at all dietary inclusion levels in F0 and F1 Cohort 1A males, which occurred concurrently with an increased but non-dose-dependent incidence of dilated renal pelvis observed macroscopically in F1 Cohort 1A males. The cause of this increased kidney weight was identified at the histopathological examination, by the presence of minimal to moderate hyaline droplet accumulation. The increase in hyaline droplets in the kidneys of males is consistent with the accumulation of α2μ‑globulin, a common finding in untreated male rats, as indeed seen in Control F1 Cohort 1A males at a low incidence. Hyaline droplet accumulation is specific to the male rat and is generally not considered to be significant in man. However, clearance or accumulation of α2μ‑globulin can be altered when it is bound to a xenobiotic, and accumulation of this complex can lead to tubular damage and this is described as α2μ‑globulin nephropathy, in this study manifest as minimal/moderate multifocal tubular basophilia and the formation of casts. Diffuse or multifocal tubular dilation was also present in males given 8000 or 16000 ppm in the F0 generation only and this indicates tubular changes associated with D,L‑Menthol; this finding is not directly associated α2μ‑globulin nephropathy and therefore may be of toxicological significance. The α2μ‑globulin is a protein synthesized by the liver of male rats only and therefore its accumulation and subsequent tubular damage is both sex and species specific. In the context of this study, the hyaline droplet accumulation and subsequent associated pathology were considered adverse in the animals affected, since degenerate changes were also present, but, due to the reasons specified previously, such findings have long been considered irrelevant in humans. These findings occurred at all administered dietary inclusion levels, and generally showed a dose-dependent increase in incidence/severity. There were some minor changes in the blood plasma of the males, with increase in urea concentrations in F0 and F1 Cohort 1A males and increase in calcium concentration in F1 Cohort 1A males, along with a slight increase in acidity of the urine of F1 Cohort 1A males that may be consequences of hyaline droplet accumulation. There was a decrease in plasma creatinine in F0 and F1 Cohort 1A females and an increase in plasma sodium and calcium concentrations in F0 females which would possibly indicate minor alterations in renal function due to excretion of the test item and/ or its metabolites, with increased kidney weights also apparent in F0 females, although there was no associated histopathological finding in these females and the subtle findings in females were therefore considered not to be adverse.

Centrilobular hypertrophy (minimal/slight) in the liver occurred at an increased incidence in F0 and F1 Cohort 1A females at all dietary inclusion levels, and correlated with a slight organ weight increase. These hypertrophic changes are suggestive of an adaptive response to mixed function oxidase induction in the liver, but the precise mechanism of hypertrophy cannot be determined by histopathological examination alone (Sahota et al, 2013). The change identified in this study was of mild or minimal

severity, limited to females and was not accompanied by other test item-related changes in the liver.

There was no histopathological correlate for the slight, but statistically significant and dose-dependent increase in liver weight consistently observed in both generations of F0 and F1 Cohort 1A males.

There was no effect of parental D,L-Menthol administration on F1 or F2 offspring birth weight, anogenital distance, sex ratio, survival after Day 1 of age to weaning or clinical condition. When compared to Controls, for F1 litters in the 16000/8000 ppm group, the mean number of uterine implantation sites was statistically significantly low, resulting in a lower total number of offspring born and subsequently live offspring on Day 1 and Day 4 of age (before litter standardization). Whilst differences in these litter

parameters in the 8000/10000/16000/8000 ppm group F2 litters did not attain statistical significance, mean values of litter size in the high dose F1 and F2 litters were lower than all other study groups in both generations, and five Control groups from three OECD 443 studies conducted during 2017-2019 (range of mean live litter size, 13.2 to 15.8 pups; mean 14.6 pups). Consequently, the lower number of uterine implantation sites and subsequently low litter size in the high dose group in both generations were considered test item-related and adverse.

In both generations, offspring growth was reduced from Day 4 of age to weaning in the high dose litters, with absolute body weight at weaning of the F1 and F2 litters on Day 21 of age being 17-18% lower than Control; the extent of the reduction in offspring growth in the high dose group in both generations was considered to be adverse. Offspring growth was also slightly low in the intermediate dose litters in both generations, from Day 7 of age in the F1 litters and from Day 14 of age in the F2 litters such that absolute body weight was 6-8% lower than Control at weaning. There findings were seen together with decreased food consumption in the dams and/or offspring and although test item-related were considered not to be adverse at the extent observed.

All F1 and F2 male offspring were assessed on Day 13 of age for the presence of nipples (areolae). In both generations, a dose dependent high incidence of male offspring in the intermediate and high dose groups showed at least one pair of areolae, with the incidence being notably higher in the F2 male offspring than in the F1 male offspring. The nipples were no longer apparent when F1 males were reassessed in Week 5 of the formal F1 generation or when F2 males were re-assessed on Day 21 of age, the retention of nipples on Day 13 of age, although clearly related to parental D,L-Menthol administration, was a transient finding with no subsequent effects on the weight/ histopathology of the male reproductive organs, fertility, mating performance or sperm analysis, and therefore not adverse.

Absolute and body weight relative spleen weights were low in F1 and F2 offspring in the intermediate and high dose groups on Day 22 of age. These differences were considered to be of no toxicological significance since there were no histopathological changes in the spleen of F0 or F1 Cohort 1A animals, or effects on spleen immunophenotyping in the F1 Cohort 1A animals. The other minor differences in unselected F1 and F2 offspring organs weights were attributable to the lower terminal body weights recorded. There were no test item-related macroscopic abnormalities detected among F1 or F2 offspring dying prematurely or among F1 or F2 offspring killed at scheduled termination.

A 3.2-day delay in attainment of balano pre-putial separation (BPS) occurred in selected F1 males in the 8000/10000/16000 ppm group, which occurred in the absence of an effect on absolute body weight on the day of attainment. Whilst a relationship to treatment for this delay cannot be excluded, BPS was achieved when comparable body weights were attained in all F1 males indicating that this observation may be secondary to reductions in body weight gain, and in the absence of subsequent effects on the weight/histopathology of the male reproductive organs, fertility, mating performance or sperm analysis, the delay in attainment of sexual maturation was considered not to be adverse.

The slight increase in ovarian follicle and corpora lutea counts in F1 Cohort 1A females given 8000/10000/16000 ppm was considered to be of no toxicological significance in view of the absence of any histopathological change in the ovaries or associated increases in litter size in the F1 Cohort 1B females.

Biochemical analysis of the plasma at scheduled termination of the F0 and F1 Cohort 1A animals revealed when compared to Controls, a slight increase in gamma glutamyl transpeptidase concentrations in high dose animals in both generations and in intermediate group F1 Cohort 1A females, low glucose concentrations in low dose F0 males and in intermediate and high dose males and females in the F0 generation, and slightly high cholesterol concentrations in high dose F1 Cohort 1A animals, and slightly low albumin/globulin ratio (due to slight changes in total protein and albumin concentrations) in high dose animals of both generations. In F0 and F1 Cohort 1A females, these

biochemical changes were considered to be related to the aforementioned hypertrophy observed in the liver at histopathological examination, and were considered to be non-adverse at the degree observed. In the F0 and F1 Cohort 1A males, there were no histopathological correlates for these minor biochemical changes, therefore these differences were considered to be of no toxicological significance.

Conclusion

The purpose of this extended one generation reproductive toxicity study was to assess the influence of D,L-Menthol, an industrial chemical, on reproductive performance when administered continuously in the diet to CD rats at dietary inclusion levels up to and including 16000 ppm. The evaluation included assessment of the integrity and performance of the adult male and female reproductive tract, and systemic toxicity in pregnant and lactating females and in young and adult offspring. In addition, an evaluation of the maturing reproductive tract and its integrity and function was performed.

Based on the results obtained in this study it was concluded that the No-Observed-Effect-Level (NOEL) for reproductive performance of the F0 and F1 Cohort 1B animals was the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females due to lower litter size observed in both generations at the high dose level, a level which was associated with reduced food consumption and body weight gain in the parental animals of both generations.

Histopathological changes in the kidney of F0 and F1 Cohort 1A males, although of no relevance to man, were adverse within the context of this study and therefore a No-Observed-Adverse-Effect-Level (NOAEL) for the kidney was not established. Aside from the kidney, the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females, based upon the impaired body weight gain at the high dose level.

The NOEL for the F1 and F2 offspring up to weaning was concluded to be the intermediate dose of 512-611 mg/kg/day due to reduced pre-weaning growth in both generations.

Toxicity to reproduction/preliminary study:

In a preliminary study the influence of D,L-Menthol on reproductive performance was examined following dietary administration to CD rats, in order to identify suitable dietary inclusion levels for investigation in an extended one-generation reproductive toxicity (OECD TG 443) study.

The purpose of this study was to assess the influence of D,L-Menthol on reproductive performance following dietary administration to CD rats, in order to identify suitable dietary inclusion levels for investigation in an extended one-generation reproductive toxicity (OECD TG 443) study.

For the F0 generation, three groups of eight male rats received D,L-Menthol orally, via the diet, at concentrations of 4000, 8000 or 16000 ppm (with corn oil added to the premix at a ratio of 5:1 test item:corn oil) for two weeks before pairing and until termination. In addition, three groups of eight female rats received D,L-Menthol orally, via the diet, at concentrations of 4000, 8000 or 16000 ppm for two weeks before pairing, throughout pairing and gestation, and at concentrations of 2000, 4000 or 8000 ppm during lactation. A similarly constituted Control group received untreated basal diet with added corn oil for the same duration. 

The F1 generation comprised eight male and eight female progeny from each group, and these animals continued to receive the treated diet, at concentrations of 4000, 8000 or 16000 ppm from Day 21 of age to Week 5 of age. 

During the study, clinical condition, body weight, food consumption, blood chemistry, biomarkers (T4) and macroscopic pathology investigations were undertaken on each generation. For F0 adults only, estrous cycles, mating performance and fertility, gestation length and parturition observations, reproductive performance and organ weights were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, and macropathology for all offspring were also assessed. 

Results:

Parental responses

The mean achieved doses for F0 males were 178, 346 and 742 mg/kg/day at 4000, 8000 and 16000 ppm, respectively. For F0 females during the 2-week pre-pairing treatment period the mean achieved doses were 240, 478 and 876 mg/kg/day and during gestation were 275, 546 and 1020 mg/kg/day at 4000, 8000 and 16000 ppm, respectively. During the first two weeks of lactation the mean achieved doses were 298, 601 and 1193 mg/kg/day at 2000, 4000 and 8000 ppm, respectively.

General clinical condition and serum T4 concentrations were unaffected by treatment.

Low mean food consumption was apparent in all groups of treated F0 animals during Day 1 to Day 4 of treatment, resulting in low mean body weight gain (males given 4000 or 8000 ppm), mean body weight stasis (females given 4000 ppm) or mean body weight loss (females given 8000 ppm and both sexes given 16000 ppm) during this period. Thereafter, the body weight performance of the F0 animals was not adversely affected by treatment.

Estrous cycle regularity, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected at all dietary inclusion levels investigated.

Biochemical analysis of the plasma at scheduled termination revealed: slightly low aspartate aminotransferase activity in all groups of treated F0 males; increased gamma glutamyl transpeptidase activity in males given 16000 ppm and all groups of treated females; increased cholesterol concentration in all groups of treated males and females; low creatinine concentrations in males given 16000 ppm, and in females given 8000/4000 or 16000/8000 ppm; increased total protein concentration (due to an increase in albumin concentration) and slightly increased calcium concentration in all groups of treated males; slightly high sodium concentrations in males given 16000 ppm.

Increased kidney and liver weights, in absolute and/or body weight relative terms, were apparent at scheduled termination in males given 16000 ppm and in females given 8000/4000 or 16000/8000 ppm. No test item-related macroscopic abnormalities were detected.

Litter responses

General clinical condition, litter size, offspring survival, sex ratio and birth weight of the F1 offspring were unaffected at all dietary inclusion levels investigated.

Body weight gain from birth to weaning of the F1offspring in litters at 16000/8000 ppm or 8000/4000 ppm was lower than Control, due to reduced body weight gain from Day 7 of age at 16000/8000 ppm and from Day 17 of age at 8000/4000 ppm; offspring body weight gain was unaffected at 4000/2000 ppm. 

Macroscopic examination of offspring that died or were killed prematurely and those at scheduledtermination, revealed no findings that could be related to D,L-Menthol.

F1 generation responses

The mean achieved doses for F1 animals receiving 4000, 8000 or 16000 ppm from weaning to scheduled termination were 541, 1021 and 2129 mg/kg/day in males and 514, 1014 and 2023 mg/kg/day in females, respectively.

There were no signs at routine physical examination that could be related to administration of D,L-Menthol.

Due to the low mean body weight gain observed prior to weaning, groupmean absolute body weight on Day 21 of age for males and females in the8000 or 16000 ppm groups was lower than Control. At 16000 ppm, group mean body weight gain from Day 21-38 of age was slightly lower than Control. With the exception of the selected F1 males given 4000 ppm, all groups of treated F1 males and females showed slightly but persistently low mean food intake when compared to Controls, most notably during Days 21 -23 of age.

Biochemical analysis of the plasma at scheduled termination revealed: slightly low aspartate aminotransferase activity in all groups of treated F1 females; increased gamma glutamyl transpeptidase activity in both sexes given 16000 ppm; increased cholesterol concentration in all groups of treated females; reduced creatinine concentrations in all groups of treated males.

No test item-related macroscopic abnormalities were detected at scheduled termination.

Conclusion

Based on the outcome of this preliminary study and the achieved doses attained, it is concluded that whilst a dietary concentration of 16000 ppm is suitable for use as the high dose level in the main extended one generation study, in order to ensure that the achieved mg/kg/day dose for the F0 females during lactation and for the selected F1 generation animals during the first few weeks after weaning does not excessively exceed the limit dose, the dietary concentrations for these study phases should be lowered by 50% (to a maximum of 8000 ppm) from those given to F0 males and F0 females before pairing and during gestation. It is also concluded that the dietary levels and subsequent achieved intakes for the selected F1 generation animals require close monitoring during the progress of the selected F1 generation, and the dietary inclusion levels increased if the overall mean achieved dose for the high dose group falls close to or below 900 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
419 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Guidline study according to OECD 443.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the EOGRTS study according OECD 443, the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females, based upon the impaired body weight gain at the high dose level.

Based on the results obtained in this study it was concluded that the No-Observed-Effect-Level (NOEL) for reproductive performance of the F0 and F1 Cohort 1B animals was the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females due to lower litter size observed in both generations at the high dose level, a level which was associated with reduced food consumption and body weight gain in the parental animals of both generations.

The NOEL for the F1 and F2 offspring up to weaning was concluded to be the intermediate dose of 512-611 mg/kg/day due to reduced pre-weaning growth in both generations.

Effects on developmental toxicity

Description of key information

Developmental toxicity/teratogenicity studies:

The administration of up to 218 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

The administration of up to 405 mg/kg (body weight) of the test material to pregnant hamsters for 5 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

The administration of up to 185 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

The administration of up to 425 mg/kg (body weight) of the test material to pregnant rabbits for 13 consecutive days had no clearly
discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable
Reason / purpose:
read-across: supporting information
Principles of method if other than guideline:
Virgin adult female albino rats (Wistar derived stock) were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water.
They were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation. Beginning on Day 6 and continuing daily through Day 15 of gestation, the females were dosed with the indicated dosage s by oral intubations. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.
Body weights were recorded on Days 0, 6, 11, 15, and 20 of gestation. All animals were observed daily for appearance and behavior with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.
On Day 20 all dams were subjected to Caesarean section under surgical anesthesia and the numbers of implantation sites, resorption sites, and live and dead fetuses were recorded. The body weights of the live pups were also recorded. The urogenital tract of each dam was examined in detail for anatomical normality.
All fetuses were examined grossly for the presence of external congenital abnormalities. One-third of the fetuses of each litter underwent detailed visceral examinations employing l0 x magnification. The remaining two-thirds were cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
not specified
Specific details on test material used for the study:
Menthol natural, brazilian (FDA 71-57).
Menthol natural, brazilian (FDA 71-57) is L-menthol (CAS 2216-51-5). L-menthol is a constituent of menthol racemic (CAS 89-78-1).
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Vehicle:
not specified
Details on mating procedure:
They were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation
Duration of treatment / exposure:
Beginning on Day 6 and continuing daily through Day 15 of gestation, the females were dosed with the indicated dosage s by oral intubations. The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.
On Day 20 all dams were subjectcd to Cacsarean section under surgical anesthesia
Frequency of treatment:
daily
Duration of test:
20 days
No. of animals per sex per dose:
22-23 animals/dose
Control animals:
other: sham treated with corn oil
Details on study design:
Sex: female
Dose descriptor:
NOEL
Effect level:
218 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: no efects observed at the highest applied dose
Abnormalities:
no effects observed
Dose descriptor:
NOAEL
Effect level:
218 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at the highest applied dose
Abnormalities:
no effects observed
Developmental effects observed:
no

All dose groups: no clearly discernible effect on nidation or on maternal or fetal survival; the number of abnormalities seen in either soft or skeletal tissues not differing from the number occuring  spontaneously in the controls.

Reproduction data:

Group:

Dose (mg/kg):

 Sham  2.18  10.15  47.05  218.0

Pregnancies

Total. No.

Died or aborted (before day 20)

To term (on day 20)

25

0

25

22

0

22

23

0

23 

23

0

23 

22

0

22 

Live Litters

Total No.*

 

25

 

22

 

23

23

22

Implant sites

total No.

Average/dam*

241 

9.64

256

11.6

266

11.6

275

12.0

250

11.4

Resorptions

Total No.*

Dams with 1 or more sites resorbed

Dams with all sites resorbed

Per cent partial resorptions

Per cent complete resorptions

5

4

-

16 .0

8

3

-

13.6

-

-

-

-

2

2

-

8.70

4

4

-

18.2

Live Fetuses

Total No..

Average/dam*

Sex ratio (M//F)

236

9.44

0.79 

248

11.3

1.07 

 

266

11.6

0.72

273

11.9

0.82

246

11.2

0.85

Dead Fetuses

Total*

Dans with 1 or more dead

Dams with all dead

Per cent partial dead

Per cent all dead

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 Average Fetus Weight, g

 3.82

 3.82

 3.82

 3.85

 3.88

* Includes only those dams examined at term.

Summary of skeletal findings*

Findings

Dose (mg/kg) 

Sham

2.18

10.15

 

47.05

218.0

 Live Fetuses Examined

(at term)

 167/25

 175/22

 183/23

 190/23

 173/22

Sternebrae

Incomplete oss.

Scrambled

Bipartite

Fused

Extra

Missing

Other

80/22

1/1

14/6

92/20

11/8

93/22

2/2

17/5

 

101/19

11/4

 

92/19

1/1

 

Ribs

Incomplete oss.

Fused/split

Wavy

Less than 12

More than 13

Other

 

25/10

3/3

 

33/13

5/3

 

1/1

19/10

2/2

 

21/10

2/2

 

16/8

3/3

Vertebrae

Incomplete oss.

Scrambled

Fused

Extra ctrs. oss.

Scoliosis

Tail defects

Other

 9/5

11/6

12/7

 

 14/7

 10/7

Skull

Incomplete closure

Missing

Craniostosis

Other

 41/16

 46/15

 63/16

 67/20

 49/17

Extremities

Incomplete oss.

Missing

Extra

 

1/1

 

 

 

 

Miscellaneous

Hyoid; missing

Hyoid; reduced

 

18/13

13/10

18/8

25/1

 

27/12

16/10

22/13

20/10

 

15/9

19/7

* Numerator = Number of fetuses affected; Denominator = Number of litters

Summary of Soft Tissue Abnormalities

 Group  Material

 Dose level

mg/kg

 Dam

 Number of

pubs

 Description
 254  FDA 71 -57  10.15

K 4044

K 4053

1

1

Petechiae

Anophthalmia

 255

  FDA 71 -57

40.05 

K 4064

K 4066

K 4072

K 4075

K 4084

1

1

1

1

Hydrocephalus: lobster claw

Anophthalmia

Anophthalmia

Gastroschisis

Gastroschisis

Executive summary:

The administration of up to 218 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly descernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable
Reason / purpose:
read-across: supporting information
Principles of method if other than guideline:
Virgin, adult, Dutch-belted female rabbits were individually housed in mesh bottom cages in temperature and humidity-controlled quarters with free access to food and fresh tap water. On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the marginal ear vein. Three hours later, each doe was inseminated artificially with 0.3 ml of diluted semen from a proven donor buck using approximately 20 x 10E6 motile sperm according to the procedure described by Vogin et al (Pharmacologist 11,282 (1969)). Beginning on Day 6 and continuing daily through Day 18 the females were dosed with the indicated dosages by oral intubation.
The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.
Body weights ware recorded on Days 0, 6, 12, 18, and 29 of gestation. All animals were observed dailly for appearance and behavior, with particular attention to food consumption and body weight in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.
On Day 29 all does were subjected to Caesarean section under surgical anesthesia, and the numbers of corpora lutea, implantation sites, resorption sites and live and dead fetuses were recorded. Body weights of the live pups were also recorded. The urogenital tract of each animal was examined in detail for normality. In addition all fetuses underwent a detailed gross examination for the presence of external congenital abnormalities. The live fetuses of each litter were then placed in an incubator for 24 hours for the evaluation of neonatal survival. All surviving pups were sacrificed, and all pups examined for visceral abnormalities (by dissection). All fetuses were then cleared in potassium hydroxide (KOH), stained with alizarin red S dye and examined for skeletal defects.
GLP compliance:
not specified
Specific details on test material used for the study:
Menthol natural, brazilian (FDA 71-57).
Menthol natural, brazilian (FDA 71-57) is L-menthol (CAS 2216-51-5). L-menthol is a constituent of menthol racemic (CAS 89-78-1).
Species:
rabbit
Strain:
Dutch
Route of administration:
oral: gavage
Vehicle:
not specified
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the marginal ear vein. Three hours later, each doe was inseminated artificially with 0.3 ml of diluted semen from a proven donor buck using approximately 20 x 10E6 motile sperm according to the procedure dcccribed by Vogin et al (Pharmacologist 11, 282 (1969)).
Duration of treatment / exposure:
Beginning on Day 6 and continuing daily through Day 18 the females were dosed with the indicated dosages by oral intubation.
The controls were sham treated with the vehicle at a level equivalent to the group receiving the highest test dose.
Body weights ware recorded on Days 0, 6, 12, 18, and 29 of gestation.
On Day 29 all does were subjected to Caesarean section under surgical anesthesia
Frequency of treatment:
daily
Duration of test:
29 days
No. of animals per sex per dose:
15 - 19 animals/dose
Control animals:
other: sham treated with corn oil
Details on study design:
Sex: female
Dose descriptor:
NOEL
Effect level:
425 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
other: no effects observed at the highest applied dose
Abnormalities:
no effects observed
Dose descriptor:
NOAEL
Effect level:
425 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed at the highest applied dose
Abnormalities:
no effects observed
Developmental effects observed:
no

All dose groups: no clearly discernible effect on nidation or on maternal or fetal survival; the number of abnormalities seen in either soft or skeletal tissues not differing from the number occuring spontaneously in the controls.

Reproduction data:

Group:

Dose (mg/kg):

 Sham  4.25  19.75  91 .7  425.0

Pregnancies

Total. No.

Died or aborted (before day 29)

To term (on day 29)

12

1

11

13

2

11

12

3

9

11

1

10 

14

4

10

Corpora Lutea

Total No.

Average/dam mated

Live litters

Total No.*

Implant sites

Total No.

Average/dam*

161

12.4

11

71 

6.45

216

14.4

10

73

6.64

165

11.8

9

64

7.11

140

12.7

10

65

6.50

213

15.2

9

72

7.20

Resorptions

Total No.*

Dams with 1 or more sites resorbed

Dams with all sites resorbed

Per cent partial resorptions

Per cent complete resorptions

10

5

-

45.5

1

1

-

9.09

5

5

-

55.6

4

4

-

40.0

11

3

1

30.0

10.0 

Live Fetuses

Total No..

Average/dam*

Sex ratio (M//F)

61

5.55

0.85 

70

6.36

1.12 

 

59

6.56

1.68

61

6.10

0.97

61

6.10

0.97

Dead Fetuses

Total*

Dans with 1 or more dead

Dams with all dead

Per cent partial dead

Per cent all dead

-

-

-

-

2

1

1

9.09

9.09

-

-

-

-

-

-

-

-

-

-

-

-

-

 Average Fetus Weight, g

 35.3

 33.5

 38.7

 36.8

 38.5

* Includes only those dams examined at term.

Summary of skeletal findings*

Findings

Dose (mg/kg) 

Sham

4.25

19.75

 

97.1

425.0

 Live Fetuses Examined

(at term)

 61/11

 70/10

 59/9

 61/10

 60/9

Sternebrae

Incomplete oss.

Scrambled

Bipartite

Fused

Extra

Missing

Other

3/2

2/1

1/1

1/1

1/1

3/2

1/1

1/1

1/1

2/1

2/1

 

1/1

1/1

 

3/3

1/1

 2/2

Ribs

Incomplete oss.

Fused/split

Wavy

Less than 12

More than 13

Other

 

 

1/1

Vertebrae

Incomplete oss.

Scrambled

Fused

Extra ctrs. oss.

Scoliosis

Tail defects

Other

 

1/1

 

 

1/1

 

1/1

2/2

1/1

Skull

Incomplete closure

Missing

Craniostosis

Other

 

 

 

 

 1/1

Extremities

Incomplete oss.

Missing

Extra

 

 

 

 

 

* Numerator = Number of fetuses affected; Denominator = Number of litters

Summary of Soft Tissue Abnormalities

 Group  Material

 Dose level

mg/kg

 Dam

 Number of

pubs

 Description
 251  Sham  0.0

S 6421

S 6424

2

1

Anopia

Umbilical hernia

 253

  FDA 71 -57

4.25 

K 6012

Umbilical hernia

 255

  FDA 71 -57

 91.7

 K 6047

 1

Medial rotation of hind limbs

 256

 FDA 71 -57

 425.0

 K 6072

 1

 Club foot; hydrocephaly

Executive summary:

The administration of up to 425 mg/kg (body weight) of the test material to pregnant rabbits for 13 consecutive days had no clearly

discernible effect on nidation or on maternal or fetal survival. The number of abnorrnalities seen in either soft or skeletal tissues of the

test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
218 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Scientifically acceptable and well documented.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Development toxicity/teratogenicity studies on rats, rabbits, mice and hamsters revealed no evidence of teratogenic effects of menthol.

Toxicity to reproduction: other studies

Description of key information

Groups of 50 rats of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm (= ca. 188 or 375 mg/kg bw/d) for 103 weeks. At the end all animals were killed and necropsied; gross and microscopic examination of: all major organs, including reproductive organs; in male rats prostate, testes, epididymis, scrotum; in female rats mammary gland, vagina, uterus, uterus/endometrium, ovary/panovarian, and ovary.

Histopathological examinations of the reproduction organs of rats showed no changes in the repeated dose toxicity studies with D/L-menthol and also in carcinogenicity studies with D/L-menthol. Hence there is no indication of a potential of D/L-menthol to interfere adversely with reproduction.

Conclusion: There is no evidence indicating a potential of D/L-menthol to interfere adversely with reproduction. Histopathological examinations of the reproduction organs of rats and mice showed no changes in repeated dose toxicity studies with D/L-menthol and also in carcinogenicity studies with D/L-menthol (OECD SIDS for methols, UNEP publication 2003).  

Recent literature (Mangelsdorf et al 2003, Ulbrich & Palmer 1995, Dent 2007, Sanbuissho et al. 2009) concluded that in rodents histopathological examinations in repeated dose toxicity studies of reproductive tissues are of high value and high sensitivity for evaluation of reproductive toxicity in males and females. Data evaluation of 117 substances or substance classes by Ulbrich & Palmer (1995)  revealed that  histopathology and organ weight analysis provide the best general purpose means of detecting substances with potential to affect male fertility, particularly those related to effects on spermatogenesis. The data evaluation by Mangelsdorf et al (2003) revealed that the most sensitive endpoint for detecting adverse effects of chemicals on male reproduction is the histopathology of the testis. Sanbuissho et al (2009) revealed that comprehensive histopathological examination of the female reproduction organs is a good tool to assess female reproduction function.  Pathological findings of ovarian toxicity (decreases in follicles, increases in atretic follicles, increases in currently formed corpora lutea, etc.) reflected the female fertility parameter (irregular estrous cycle, pre-implantation loss). The data analysis by Dent (2007) revealed that subchronic toxicity studies are suitable to predict effects on rodent fertility.  - Mangelsdorf. et al., 2003: Some aspects relating to the evaluation of the effects of chemicals on male fertility. Regulatory toxicology and Pharmacology 36, 69-98 - Ulbrich & Palmer, 1995: Detection of effects on male reproduction – a literature survey. J Am. College of Toxicology 14, 293-327 - Janer et al., 2007: A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study. Reproductive Toxicology 24, 103-113 - Dent, 2007: Strength and limitations of using repeated-dose toxicity studies to predict effects on fertility. Regulatory Toxicology and Pharmacology 48, 241-258 - Sanbuissho et al., 2009: Collaborative work on evaluation of ovarian toxicity by repeated-dose and fertility studies in female rats. J Tox. Sci. 34: Special Issue SP1-SP22 - Piersma et al., 2011: Combined retrospective analysis of 498 rat multi-generation reproductive toxicity studies: on the impact of parameters related to F1 mating and F2 offspring. Reproductive Toxicology 31, 392-401.  

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction: other studies
Remarks:
other: carcinogenicity study with histopathologic examiniation of male and female reproduction organs
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: other: carcinogenicity study with histopathologic examiniation of male and female reproduction organs
Principles of method if other than guideline:
A bioassay of dl-menthol for possible carcinogenicity was conducted by administrating the test chemical in feed to Fisher 344 rats.
Groups of 50 rats of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm for 103 weeks, then observed for 1 or 2 additional weeks. Matched controls consisted of 50 untreated rats of each sex. All surviving rats were killed at 105 weeks.
At the end all animals were killed and necropsied; gross and microscopic examination of: all major organs, including reproductive organs; in male rats prostate,testes, epididymis, scrotum; in female rats mammary gland, vagina, uterus, uterus/endometrium, ovary/panovarian, ovary.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2 batches test:
Lot No. 4-HTP-6
Lot No. Nl 1-26-74-2054

Identity confirmed
Batch 1:
2 impurites:
0.3 %
1.3 %
Batch 2:
0.2 % Impurity
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
daily
Dose / conc.:
3 570 ppm
Remarks:
ca. 188 mg/kg bw/d
Dose / conc.:
7 500 ppm
Remarks:
ca. 375 mg/kg bw/d
No. of animals per sex per dose:
50 rats of either sex/dose
Control animals:
other: untreated diet containing 2 % corn oil
Dose descriptor:
NOAEL
Effect level:
ca. 375 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 375 mg/kg bw/d was the highest applied dose
Conclusions:
Histopathological examinations of the reproduction organs of rats and mice showed no changes in repeated dose toxicity studies with D/L-menthol and also in carcinogenicity studies with D/Lmenthol.
Hence there is no indication of a potential of D/L-menthol to interfere adversely with reproduction.
Executive summary:

Groups of 50 rats of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm for 103 weeks, then observed for 1 or 2 additional weeks. Matched controls consisted of 50 untreated rats of each sex. All surviving rats were killed at 105 weeks. Body weights and clinical signs were recorded and pathological and histopathological examinations conducted. At the end all animals were killed and necropsied; gross and microscopic examination of: all major organs, including reproductive organs; in male rats prostate,testes, epididymis, scrotum; in female rats mammary gland, vagina, uterus, uterus/endometrium, ovary/panovarian, ovary.

Histopathological examinations of the reproduction organs of rats showed no changes in the repeated dose toxicity studies with D/L-menthol and also in carcinogenicity studies with D/Lmenthol. Hence there is no indication of a potential of D/L-menthol to interfere adversely with reproduction.

Endpoint:
toxicity to reproduction: other studies
Remarks:
other: carcinogenicity study with histopathologic examiniation of male and female reproduction organs
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: other: carcinogenicity study with histopathologic examiniation of male and female reproduction organs
Principles of method if other than guideline:
A bioassay of dl-menthol for possible carcinogenicity was conducted by administrating the test chemical in feed to B6C3F1 mice.
Groups of 50 mice of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm for 103 weeks, then observed for 1 or 2 additional weeks. Matched controls consisted of 50 untreated mice of each sex. All surviving mice were killed at 104 weeks.
At the end all animals were killed and necropsied; gross and microscopic examination of: all major organs, including reproductive organs; in male mice penis, prepuce, preputial gland, prostate, epididymis; in female mice uterus, uterus/endometrium, ovary.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2 batches test:
Lot No. 4-HTP-6
Lot No. Nl 1-26-74-2054

Identity confirmed
Batch 1:
2 impurites:
0.3 %
1.3 %
Batch 2:
0.2 % Impurity
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
103 w
Frequency of treatment:
daily
Dose / conc.:
2 000 ppm
Remarks:
ca. 334 mg/kg bw/d
Dose / conc.:
4 000 ppm
Remarks:
ca. 667 mg/kg bw/d
No. of animals per sex per dose:
50 mice of each sex per dose
Control animals:
other: untreated diet containing 2 % corn oil
Dose descriptor:
NOAEL
Effect level:
ca. 667 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 667 mg/kg bw/d was the highest applied dose
Conclusions:
Histopathological examinations of the reproduction organs of rats and mice showed no changes in repeated dose toxicity studies with D/L-menthol and also in carcinogenicity studies with D/Lmenthol.
Hence there is no indication of a potential of D/L-menthol to interfere adversely with reproduction.
Executive summary:

Groups of 50 mice of each sex were administrated dl-menthol at one of the following doses, either 3750 or 7500 ppm (= ca. 334 or 667 mg/kg bw/d) for 103 weeks, then observed for 1 or 2 additional weeks. Matched controls consisted of 50 untreated mice of each sex. All surviving mice were killed at 104 weeks. At the end all animals were killed and necropsied; gross and microscopic examination of: all major organs, including reproductive organs; in male mice penis, prepuce, preputial gland, prostate, epididymis; in female mice uterus, uterus/endometrium, ovary.

Histopathological examinations of the reproduction organs of mice showed no changes in the repeated dose toxicity studies with D/L-menthol and also in carcinogenicity studies with D/Lmenthol. Hence there is no indication of a potential of D/L-menthol to interfere adversely with reproduction.

Additional information

No data.

Justification for classification or non-classification

Fertility study:

In the EOGRTS study according OECD 443, the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females, based upon the impaired body weight gain at the high dose level.

Based on the results obtained in this study it was concluded that the No-Observed-Effect-Level (NOEL) for reproductive performance of the F0 and F1 Cohort 1B animals was the intermediate dose of 419-499 mg/kg/day for males and 455-594 mg/kg/day for females due to lower litter size observed in both generations at the high dose level, a level which was associated with reduced food consumption and body weight gain in the parental animals of both generations.

The NOEL for the F1 and F2 offspring up to weaning was concluded to be the intermediate dose of 512-611 mg/kg/day due to reduced pre-weaning growth in both generations.

Developmental toxicity/teratogenicity studies:

The administration of up to 218 mg/kg (body weight) of the test material to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

The administration of up to 185 mg/kg (body weight) of the test material to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

The administration of up to 405 mg/kg (body weight) of the test material to pregnant hamsters for 5 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

The administration of up to 425 mg/kg (body weight) of the test material to pregnant rabbits for 13 consecutive days had no clearly
discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.

Based on the results of the EOGRTSand the developmental toxicity studies a classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is not justified.