o-cresol

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: TSCA Health Effects Test Guideline for specific organ/tissue toxicity - Reproduction/Fertility effects (EPA, 1983)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

o-cresol

EC Number:

202-423-8

EC Name:

o-cresol

Cas Number:

95-48-7

Molecular formula:

C7H8O

IUPAC Name:

o-cresol

Details on test material:

Test substance: o-cresol, purity: 99.7 %

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: 6 wks (P) ;
- Weight at study initiation: (P) Males: 193-194g; Females: ca. 151 g;
- Housing: initially 2/same sex during acclimation period; and then singly except for the cohabitation and lactation periods
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature :68-74°F
- Humidity (%): 40-60
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared by weighing the amount of test chemical into a volumetric flask and diluting to volume with certified corn oil.
The resulting solutions were mixed by repeated inversions and stored at room temperature.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged singly
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A standard stock solution was prepared (1mg/ml propanol),which was used to prepare standards ranging fron 10 to 100 ng/µl. With these solutiower a standard curve was generated using HPLC. Dosing formulation concentrations were veryfied by preparing aliquots which were injected onto the HPLC column. The measured concentration of each dosing solution was then calculated from the equitation for the standard curve developed by linear regression.

Duration of treatment / exposure:

Exposure period: 27 w
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks

Frequency of treatment:

once per day , 5 d/w
F1 generation producing F2: once per day, 7 days per week

Details on study schedule:

at day 28-40 post partum F1 animals selected to be parents of the F2 generation were gavaged with their respective formulations for at least
11 weeks 5 days/week.
the F1 animals were approximately 15 to 17 weeks of age at the initiation of the mating period.
They were dosed from that time point 7 days/week. Mating procedure was performed as done with the P-generation

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 175, 450 mg/kg/day in corn oil
Basis:
actual ingested

No. of animals per sex per dose:

25 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

no further data

Positive control:

no data

Examinations

Parental animals: Observations and examinations:

Mortality: twice daily
General conditon: daily throughout the course of the study including skin and fur, eyes and mucous membranes, respiratory symptoms, circulatory system, autonomic and central nervous system, somatomotor activity, behavior pattern.
Body weight determination
male, female: initially and weekly until mating
female during gestation : day 0, 7, 13, 20 post partum: day 0, 4, 7, 14, 21
Food consumption
measured weekly during pre-breed dosing period for P and F1 generation; all other phases of this sutdy determination was made visually

Oestrous cyclicity (parental animals):

Vaginal smears were examined to determe pregnancy.

Sperm parameters (parental animals):

no data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the completion of the mating period
- Maternal animals: All surviving animals after the F1 and F2 litters have been weaned


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
A complete gross necropsy was conducted for any parental animal dying on test.


HISTOPATHOLOGY
Male and female adult rats of the highest dose groups and the controls
The tissues as indicated below, were prepared for microscopic examination and weighed, respectively:
pituitary, vagina, uterus, ovaries, testes, epididymides, seminal vesicles, prostate and other tissues with gross lesions identified as being
potentially treatment-related.
A complete histopathological examination was conducted for any parental animal dying on test.

Postmortem examinations (offspring):

All pups dying during lactation are neccropsied to investigate the cause of death.

At weaning , postnatal day 21 , 1 female and 1 male from each F1 litter is selected on a random basis to become parents of the next
generation.
The remaining offspring is cexamined for gross external abnormalities , euthanized and discarded.

Statistics:

Levene's test for equal variances, analysis of variance (ANOVA), t-test, Kruskal-Wallis test, Mann-Whitney U test Fisher's exact test

Reproductive indices:

calculated for P and F1 animals:
Mating Index (%):
---for males: number of males impregnating females devided through the total number of males paired multiplied by 100
---for females: number of females with copulation plugs devided through the mumger of females cohabited multiplied by 100

Fertility Index(%):
---for males: number of males producing pregnant females devided through number of males impregnating females multiplied by 100
---for females. number of pregnant females devided through number of plug-positive females multiplied by 100

Gestational Index (%)
number of females with live litters devided through number of females pregnant multiplied by 100

Offspring viability indices:

calculated for F1 and for F2 animals
live birth index (%)
number of live pups at birth devided through the toal number of pups born multiplied by 100
4-Day Survival Index (%):
number of pups surviving 4 days devided through total number of live pups at birth multiplied by 100
7-Day Survival Index(%):
number of pups surviving 7 days devided through total number of live pups at 4 days multiplied by 100
14- Day Survival Index (%):
number of pups surviving 14 days devided through total number of live pups at 7 days multiplied by 100
21-day survival index (%):
number of pups surviving 21 days devided through total number of live pups at 14 days multiplied by 100
Lactation index (%):
number of pups surviving 21 days devided through total number of live pups at 4 days multiplied by 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not specified

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

see section "remarks on results"

Effect levels (P0)

1

Dose descriptor:

other: NOAEL (fertility)

Effect level:

450 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: There were no treatment related reproductive effects observed in this study.

Remarks on result:

other: Generation: F0 and F1 (parental)

2

Dose descriptor:

other: NOAEL (offspring)

Effect level:

175 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: F1 (males) significantly reduced body weights at 450 mg/kg bw group F2: Significantly reduced lactation index in the 450 mg/kg bw group.

Remarks on result:

other: Generation: F1 and F2

3

Dose descriptor:

other: NOAEL (general toxicity)

Effect level:

30 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: 450mg/kg bw: increased mortaliity; 450 and 175 mg/kg bw/d: Signs of intoxication

Remarks on result:

other: Generation: F0 and F1

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

175 mg/kg bw/day (nominal)

System:

other:

Organ:

other: 450mg/kg bw: increased mortaliity; 450 and 175 mg/kg bw/d: Signs of intoxication

Treatment related:

yes

Dose response relationship:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Details on results (F1)

see section " remarks on results"

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

175 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: F1 (males) significantly reduced body weights at 450 mg/kg bw group F2: Significantly reduced lactation index in the 450 mg/kg bw group

Remarks on result:

other: Generation: F1 and F2

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

Organ:

other: F1 (males) significantly reduced body weights at 450 mg/kg bw group F2: Significantly reduced lactation index in the 450 mg/kg bw group.

Treatment related:

yes

Dose response relationship:

yes

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

175 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: F1 (males) significantly reduced body weights at 450 mg/kg bw group F2: Significantly reduced lactation index in the 450 mg/kg bw group.

Target system / organ toxicity (F2)

Critical effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (nominal)

Organ:

other: F1 (males) significantly reduced body weights at 450 mg/kg bw group F2: Significantly reduced lactation index in the 450 mg/kg bw group.

Treatment related:

yes

Dose response relationship:

yes

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

F0-generation:
---Mortality during pre-breed period:
450 mg/kg bw/d males: 12/25 and females: 8/25
---Signs of intoxication.
450 mg/kg bw/d, males and females:
hypoactivity, ataxia, twitches, tremors, prostration, gasping, rapid respiration, lacrimation, amd perioral wetness occurred immediately after dosing but did not persist until the next day.
---Mean weight gain in F0 males and F0 females were equivalent across the groups
---Mean body weight, significantly reduced: males: 175 mg-gr., 450 mg-gr., week 13: 489.9 g,470.9 g versus 522 g of controls.

---Reproductive parameters for F0 parents at F1 breed:
Mating index (males and females), fertility index (males and females) and gestational index were not affected by treatment.
--Body weight development during gestation and lactation was equivalent across the groups.
--F1-pups:
-Live-birth index was comparable across the groups, sex ratio was not affected
-Survival Indices: 4-day-, 7-day-, 14-day- and 21-day-survival indices and lactation index were equivalent across the groups.
450 mg/kg bw/d,males (pre-breed period): significantly reduced mean body weights: 139.6 g versus 162 g in controls
F1-adult-generation
---Signs of intoxication.
450 mg/kg bw/d, males and females: hypoactivity, ataxia, twitches, tremors, prostration, gasping, rapid respiration, lacrimation, and perioral wetness.
175 mg/kg bw/d:
males: perioral wetness
females: hypoactivity, ataxia, perioral wetness
---Mortality
450 mg-group:
pre-breed period: 8 males and 14 females
additionally:
7 males and 9 females died prior to mating, 3 females which were found pregnant died and another one died during gestation
---body weight and weight development
females: equivalent across the groups
males: week 14, low, mid, high dose: 586.6 g, 564.1 g, 494.4 g versus 526.3 g of controls
---Reproductive parameters for F1 parents at F2 breed:
Mating index (males and females), fertility index (males and females) and gestational index were not affected by treatment.
--Body weight development during gestation and lactation was equivalent across the groups.
--F2-pups:
-Live-birth index was comparable across the groups, sex ratio was not affected
-Survival Indices: 4-day-, 7-day-, 14-day- and 21-day-survival indices were equivalent across the groups.
lactation index
450 mg-group: was significantly reduced (75.5 versus 99.4 in controls)

Conclusion:
The A/D ratio (the dose level at which there were no observable effects in offsprings) is less than 1: (30/175) indicating no increased risk to offspring from o-cresol in the absence of parental effects.

Applicant's summary and conclusion

Executive summary:

Two-generation reproductive toxicity according to TSCA Health Effects Test Guideline for specific organ/tissue toxicity - Reproduction/Fertility effects (EPA, 1983)

Continued administration of o-cresol by gavage for two generations to Sprague-Dawley rats resulted in general toxicity including increased mortality at 450 mg/kg bw/d and in clinical signs of toxicity including hypoactivity, ataxia, twitches, tremors, prostration, gasping, rapid respiration and perioral wetness at >= 175 mg/kg bw/d (NOAEL (genral toxicity) 30 mg/kg bw/d). No reproductive parameters were affected by treatment in either of the two generations (NOAEL (fertility) 450 mg/kg bw/d.

The NOAEL (offspring) is 175 mg/kg bw/d based on significantly reduced body weights in F1 males and in significantly reduced lactation index in F2 at 450 mg/kg bw.