4-methylanisole

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The test compound did not adversely affect fertility of the F0 generation parental animals at all dose levels as there were no changes of male/female mating and fertility indices, time until successful copulation, duration of pregnancy and mean number of implantations.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to GLP and current testing guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: 11 - 13 wks
- Weight at study initiation: male animals: 281.6 g - 321.8 g; female animals: 170.3 g - 206.8 g
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), except for overnight matings and females with litters. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added.
- Diet : ad libitum; ground Kliba maintenance diet mouse/rat “GLP” meal (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 21 Dec 2009- 09 Feb 2010 (administration period)

Route of administration:

oral: gavage

Vehicle:

olive oil

Remarks:

Ph.Eur./DAB

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in Olive oil Ph.Eur./DAB were prepared at the beginning of the
administration period. The maximum period for which each preparation was used was 7 days.
For the preparation of the administration solutions the test substance was weighed in a
graduated measuring flask depending on the dose group, topped up with Olive oil
Ph.Eur./DAB and subsequently thoroughly shaken until completely dissolved.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 2.5; 7.5; 25 g/100 ml
- Amount of vehicle (if gavage): 4 ml/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1 / 1 ratio
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes and in females the entire gestation period as well as
4 days of lactation and approximately 1 week thereafter.

Frequency of treatment:

once daily

Details on study schedule:

Males and females from the same dose group were mated 13 days after the beginning of treatment.

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: On the basis of data from available repeateded dose studies

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: once a week until sacrifice
During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
Food consumption was not determined in females without positive evidence of sperm during gestation periods and in females without litter during lactation period.
Food consumption was not determined after 2nd premating week and during the mating period
Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 and 4.

Oestrous cyclicity (parental animals):

not assessed

Sperm parameters (parental animals):

not assessed

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups (Sex ratio), stillbirths, live births, postnatal mortality, presence of gross anomalies, clinical symptoms, body weights


GROSS EXAMINATION OF DEAD PUPS:
Pups were examined externally, eviscerated and their organs were assessed macroscopically.

Postmortem examinations (parental animals):

SACRIFICE
Seven days after PND 4 of the female, which delivered last, all parental females were sacrificed and examined.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations (special attention given to the reproductive organs).

HISTOPATHOLOGY / ORGAN WEIGHTS
Epididymides, testes, ovaries were weighed.
Histopathology was performed for all gross lesions (all dose groups), epididymides, testes, ovaries (high dose group and controls)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
Pups were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

DUNNETT-test:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter.
FISHER'S EXACT test:
Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy.
WILCOXON-test:
Proportions of affected pups per litter with necropsy observations
KRUSKAL WALLIS test + WILCOXON-test (Postest):
Weight parameters

Reproductive indices:

Male/female mating index, Male/female fertility index, Gestation index, Live birth index, Postimplantation loss

Offspring viability indices:

Viability index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day:
Abdominal position after treatment during treatment week 0 (all males and females), week 1 (2 males and 2 females) and week 6 (1 female); and during GD 15, 18 or 21 (2 females)
• Apathy after treatment during treatment week 0 (5 males and 7 females) and week 1 (one male)
• Ataxia after treatment during treatment week 0 (3 males) and weeks 0 and 1 (8 and 7 females, respectively)
• Unsteady gait after treatment during treatment week 6 (3 females), GD 24 (1 female) and PND 0-4 (up to 3 females per day, 5 females in total)
• Total litter loss in all females
• Insufficient maternal care in 33% females (up to 2 of 9 females per day between PND 0 –2 having pups with no or less milk in stomach)
At 300 mg/kg/day:
• Total litter loss in 22% of females (vs. 0% in control)
• Insufficient maternal care in 44% females on day 0 of lactation (pups had no or less milk in stomach, vs. 0% in control)
At 100 mg/kg/day:
No test substance-related findings.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities in any of the male and female F0 parental animals in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of the high-dose males (1000 mg/kg bw/d) were statistically significantly lower on weeks 3 and 4 (up to -7%) and the body weight change was statistically significantly decreased between weeks 0 - 1 and 2 - 3 (up to -72%). If calculated for the entire treatment period (weeks 0 - 4) the body weight change was statistically significantly decreased 39% below control. Mean body weights and body weight change in parental males in mid and low dose groups (300 and 100 mg/kg/day) were similar to controls throughout the study.

Mean body weights of the high-dose females were statistically significantly decreased on gestation day 20 (-10%) and body weight gain was statistically significantly decreased between gestation days 0 - 20 (up to -36%). During lactation, the mean body weights of the high-dose females were statistically significantly decreased on day 4 (-10%) and body weight gain (PND 0 - 4) was statistically significantly decreased (-70%). Mean body weights and body weight changes of the high-dose females during premating were comparable to the concurrent control group. The body weights and body weight changes of the low- and mid-dose F0 females were comparable to the concurrent control group throughout the entire study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high-dose F0 males (1000 mg/kg bw/d) was statistically significantly below control during premating weeks 0 - 1 (-10%). During premating weeks 1 - 2 food consumption of the high- and mid-dose F0 males (1000 or 300 mg/kg bw/d) was statistically significantly above control (18 or 8%). Low-dose males (100 mg/kg bw/d) did not show any test substance-related changes of food consumption during the whole treatment period.

Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was statistically significantly below control during premating weeks 0 - 1 (-16%). During premating weeks 1 - 2 food consumption of the high- and mid-dose F0 females (1000 or 300 mg/kg bw/d) was statistically significantly above control (25 or 16%). During gestation, food consumption of the high- and mid-dose F0 females was statistically significantly above control between GD 0 - 14 (test group 3, up to 23%) or GD 0-20 (test group 2, up to 17%). During lactation (PND 1-4), food consumption of the high- and mid-dose F0 females (1000 or 300 mg/kg bw/d) was statistically significantly below control (test group 3 -52%, test group 2 - 33%).

During lactation (PND 1-4), food consumption of the high- and mid-dose F0 females (1000 or 300 mg/kg bw/d) was statistically significantly below control (test group 3 -52%, test group 2 - 33%).

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

the male mating index was 100% in all groups including the controls.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The fertility index varied between 80% (test group 1), 90% (test groups 2 and 3) and 100% (control). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The gestation index was 100% in all groups. The incidence of litters with stillborn pups was statistically significantly increased at 1000 and 300 mg/kg bw/d being 100% (p≤0.01) and 44% (p≤0.05), respectively.
The rate of liveborn pups was statistically significantly reduced in test group 3 (76, p≤0.01) and test group 2 (113, p≤0.01) vs. 121 in control), as indicated by live birth indices of 84% (test group 3) and 90% (test group 2) vs. 100% in control and test group 1. The number of stillborn pups was statistically significantly increased in test group 3 (16%, p≤0.01) and test group 2 (9.6%, p≤0.01). In the low-dose group (100 mg/kg bw/d) the rate of liveborn and stillborn pups was comparable to the control.

The male fertility index ranged between 80% and 100% without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

All high- and mid-dose females (GD 0 - 25) and six low-dose females (GD 0 - 25) showed salivation after treatment. This transient salivation for a few minutes immediately after each treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity nor as adverse.

1000 mg/kg bw/day:
• Abdominal position after treatment during treatment week 0 (all males and females), week 1 (2 males and 2 females) and week 6 (1 female); and during GD 15, 18 or 21 (2 females)
• Apathy after treatment during treatment week 0 (5 males and 7 females) and week 1 (one male)
• Ataxia after treatment during treatment week 0 (3 males) and weeks 0 and 1 (8 and 7 females, respectively)
• Unsteady gait after treatment during treatment week 6 (3 females), GD 24 (1 female) and PND 0-4 (up to 3 females per day, 5 females in total)
• Total litter loss in all females
• Insufficient maternal care in 33% females (3 of 9 females between PND 0 – 2 having pups with no or less milk in stomach)
• Decreased food consumption in males (-10%) and females (-16%) during treatment weeks 0 – 1 and in females during lactation (-52%, PND 1-4)
• Increased food consumption in males (18%) and females (25%) during treatment weeks 1 – 2 and GD 0 – 14 (up to 23%)
• Decreased body weights in males in treatment weeks 3 and 4 (up to -7%) and in females on GD 20 (-10%) and PND 4 (-10%)
• Decreased body weight gain in males in treatment weeks 0 – 1 and 2 - 3 (up to -72%) and weeks 0 – 4 (-39%); in females between GD0–20 (up to-36%) and PND 0-4 (-70%)
• Increased incidence of postimplantation loss (17.4% vs. 6.3% in control)
• Increased number of litters with stillborn pups (100% vs. 0% in control)
• Reduced live birth index (84% vs. 100% in control),
• Increased number of stillborn pups (16% vs. 0% in control)
• Decrease of terminal body weight in males (-9%) and females (-8%)
• Enlarged livers characterized as centrilobular hypertrophy of hepatocytes in all males and females

300 mg/kg bw/day:
• Total litter loss in 22% of females (vs. 0% in control)
• Insufficient maternal care in 44% females on day 0 of lactation (pups had no or less milk in stomach, vs. 0% in control)
• Decreased food consumption in females during lactation (-33%, PND 1-4)
• Increased food consumption in males (8%) and females (16%) during treatment weeks 1 – 2 and GD 0 – 20 (up to 17%)
• Increased number of litters with stillborn pups (44% vs. 0% in control)
• Reduced live birth index (90% vs. 100% in control)
• Increased number of stillborn pups (9.6% vs. 0% in control)
• Decrease of terminal body weight in males (-5%)
• Enlarged livers in single male (1 of 10) and females (2 of 10) partially characterized as centrilobular hypertrophy of hepatocytes

100 mg/kg bw/day:
• No test substance-related adverse findings

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

At 300 and 1000 mg/kg/day, male and female test animals showed a dose-dependent enlargement of the liver characterized by a centrilobular hypertrophy of hepatocytes, indicative for enzyme induction (cytochrome P450).

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

>= 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No test substance related adverse findings

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In test group 3 (1000 mg/kg bw/d) all pups died or were cannibalized until PND 4. Consequently, the viability index indicating pup mortality during lactation (PND 0 - 4) was 0%. In test group 2 (300 mg/kg bw/d) the viability index was distinctly reduced (58%, p≤0.01), resulting from significantly higher numbers of died (25%, p≤0.01) and cannibalized pups (14%, p≤0.01). In test group 1 (100 mg/kg bw/d) and control the viability index was 100%.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In test group 3 (1000 mg/kg bw/d), there was only one female pup alive on PND 1, which weighed about 29% less than the control. Mean body weights of the mid-dose pups were statistically significantly below control on PND 1 and PND 4. The average difference to the control was -16% on PND 1 and PND 4 (up to -17%). The mid-dose pups gained about 17% less weight than the controls, but being statistically significant only in the females and males+females (p≤0.05), respectively. No statistically significant changes on F1 pup body weights and body weight gain were observed in the low-dose group (100 mg/kg bw/d). The number of "runts" was increased in the mid dose group (10 vs. 1 in control).

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Empty stomach, which was found in 15% of the high-dose pups and in 20% of the middose pups, was considered to be treatment-related. A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and hemorrhagic testis.

1000 mg/kg bw/day:
• All pups died or were cannibalized until PND 4 (viability index = 0%)
• Decreased pup body weights on PND 1 (only one female pup alive), 29% below control
• Empty stomach in 15% of the pups at necropsy (vs. 0% in control)

300 mg/kg bw/day:
• Reduced viability index (58% vs. 100% in control), resulting from significantly higher numbers of died (25%) and cannibalized (14%) pups.
• Decreased pup body weights on PND 1 and PND 4 (up to -17%)
• Decreased pup body weight gain during PND 1 - 4 (-17%) in female as well as in male+female pups
• Higher number of runts (10 vs. 1 in control)
• Empty stomach in 20% of the pups at necropsy (vs. 0% in control)

100 mg/kg bw/day:
• No test substance-related adverse findings

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Based on pre/postnatal mortality, decreased pup weight/gain

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Conclusions:

p-Cresolmethylether was given daily as an oily solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (Olive oil
Ph.Eur./DAB). The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, and in females the entire gestation period as well as 4 days of lactation and approximately 1 week thereafter. There were no treatment related effects on reproduction. Effects at the mid and high dose related to changes in body weight gain and altered food consumption were treatment-related and were at least partially related to the poor survival of pups. Therefore, the NOAEL for reproduction is 1000 mg/kg/day, parental NOAEL and developmental NOAEL is 100 mg/kg/day.

Executive summary:

A reproduction/developmental toxicity screening test had been performed for p-Cresolmethylether. p-Cresolmethylether was given daily as an oily solution to groups of 10 male and 10 female Wistar rats (F0 animals) by stomach tube at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, and in females the entire gestation period as well as 4 days of lactation and approximately 1 week thereafter. Clinical observations indicated distinct toxicity in the exposed parental animals of the highdose and mid-dose group (1000 and 300 mg/kg bw/d) but not in the animals of the low-dose group (100 mg/kg bw/d). The test compound did not adversely affect fertility of the F0 generation parental animals at all dose levels as there were no changes of male/female mating and fertility indices, time until successful copulation, duration of pregnancy and mean number of implantations. However, there is evidence for a dose-dependent adverse effect of the test substance on pre-/postnatal development of the F1 offspring at mid and high-dose level (300 and 1000 mg/kg bw/d). At the high dose level, this was indicated by an increased postimplantation loss, decreased number of delivered/liveborn, increased number of stillborn, and a total litter loss of all females. At the mid dose, similar effects were noted. Decreased terminal body weight in males at 300 and 1000 mg/kg bw/d as well as in females at 1000 mg/kg bw/d is considered treatment-related. under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for fertility is 1000 mg/kg body weight/day for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance is 100 mg/kg body weight/day for the F0 parental animals based on abdominal position shortly after treatment, apathy, unsteady gait/ataxia, reduced food consumption and decreased body weight/body weight gain in both sexes at 1000 mg/kg body weight/day and altered food consumption in both sexes as well as decreased terminal body weight in males at 300 mg/kg body weight/day. The NOAEL for developmental toxicity is 100 mg/kg body weight/day, based on prepostnatal offspring mortality at 300 and 1000 mg/kg body weight/day as well as reduced pup weight/pup weight gain at 300 mg/kg body weight/day. The postnatal effects were at least partially secondary to disturbed maternal care.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Decrease of terminal body weight in males at 300 and 1000 mg/kg bw/d as well as in females at 1000 mg/kg bw/d is considered treatment-related. Additionally, male and female test animals showed a dose-dependent enlargement of the liver characterized by a centrilobular hypertrophy of hepatocytes, indicative for enzyme induction (cytochrome P450). The test substance led to no treatment-related changes in the genital organs of males (testes and epididymides) and females (ovaries). These results confirmed that adverse effects of the test substance on fertility were not observed in this study.

Effects on developmental toxicity

Description of key information

There is evidence for a dose-dependent adverse effect of the test substance on pre-/postnatal development of the F1 offspring at mid and high-dose level (300 and 1000 mg/kg bw/d). At the high dose level, this was indicated by an increased postimplantation loss (17.4%), decreased number of delivered/liveborn (84%), an increased number of stillborn pups (16%), and a total litter loss of all females. At the mid dose level, the same effects were noted, but at a lower incidence in live birth index (90%), increased number of stillborn (9.6%), and decreased viability index of 58%. In contrast to the high dose group, decreased pup weight / pup weight gain could be determined based on the higher number of surviving pups. At least partially, the reduced pup survival may be secondary to a disturbance of maternal care as it became obvious by empty stomachs in pups which have been observed in 20% of mid-dose and 15% of high-dose offspring.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available