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REACH

2,4,6-trimethylphenol

EC number: 208-419-2 | CAS number: 527-60-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Effects on fertility

Description of key information

Combined Repeated Dose with Reproduction/Developmental Toxicity Screening Study (OECD TG 422), rat (m/f), 2005: NOAELsystemic ≥ 200 mg/kg bw/day (highest dose tested); NOAELreproductive/developmental ≥ 200 mg/kg bw/day (highest dose tested)
Combined Repeated Dose with Reproduction/Developmental Toxicity Screening Study (OECD TG 422), rat (m/f), 2007: NOAELlocal = 10 mg/kg bw/day (lowest dose tested); NOAELreproductive/developmental ≥ 300 mg/kg bw/day (highest dose tested)

Link to relevant study records

Referenceopen all close all

Reference 1

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:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:: yes
Remarks:: , F1 offspring was followed to adulthood with continued exposure and assessment of neurologic, immunologic, and reproductive structures and functions. In addition, F0 recovery males and 28-day recovery females was assessed.
GLP compliance:: yes
Limit test:: no
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, Raleigh, NC
- Age at study initiation: ca. 63 days old
- Weight at study initiation: 276-300 g for males; 201-225 g for females
- Fasting period before study: not performed
- Housing: individually upon arrival, during the acclimation period , and upon the initiation of the treatment period in solid-bottom polycarbonate cages (8” x 19”x 10.5” high) with stainless-steel wire lids (Laboratory Products, Rochelle Park, NJ) with Sani-Chip® cage litter (P.J. Murphy Forest Products Corp., Montville, NJ. Study animals were housed 2 per cage (1 male: 1 female from the same group) during the mating period. Females were caged individually once they were successfully mated (or at the end of the mating period) and throughout gestation. Females were housed with their litters throughout the lactation period. Randomly selected F1 weanlings, males, and females were singly housed during the postweaning exposure period.
- Diet: Pelleted Purina Certified Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: Tap water (City of Durham, Department of Water Resources, Durham, NC), ad libitum, in plastics water bottles
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air: air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:: oral: gavage
Vehicle:: corn oil
Remarks:: Mazola®
Details on exposure:: VEHICLE
- Concentration in vehicle: 2, 20 and 40 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:: Following the 2-week prebreed exposure, the animals were mated on the basis of 1 male to 1 female, selected randomly within each dose group for a period of 14 days, with no change in mating partners. The observation of vaginal sperm of copulation plug was considered evidence of successful mating. Females were examined daily during the cohabitation period for the presence for sperm or copulation plug in the vaginal tract. The day vaginal sperm (or plug) were observed was designated as gd 0. Once vaginal sperm (or plug) were observed, the male and female from the mating pair were individually housed.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The analyses were carried out at the RTI International, Center for Life Science and Toxicology, 3040 Cornwallis Road, Research Traingle Park, NC 27709-2194. No problems were experienced in the preparations of the formulation of test chemical in corn oil at 2 and 40 mg/mL. The storage stability studies showed that the test chemical to be stable for 35 days when stored in amber bottles, protected from light, either at ambient temperature or in a refrigerator.
Duration of treatment / exposure:: - Experimental group, F0: 2 weeks of prebreed, 2 weeks of mating, and 3 weeks of gestation and lactation until necropsy (at least 4 weeks for males and 10 weeks for females)
- Any female that did not show evidence of successful mating after 14 days of cohabitation received continued treatment until gd 26 or delivery occurred.
- Recovery group and 28 days females: 28 days
- Selected F1 offspring: from pnd 22 (day after weaning) until necropsy (at least 7 weeks) at least 1 female and 1 male from each F1 litter, for a total of 10/sex/group, were selected on a random basis to continue treatment.
Frequency of treatment:: daily
Remarks:: Doses / Concentrations:
0, 10, 100, 200 mg/kg/day (experimental group)
Basis:
actual ingested
Remarks:: Doses / Concentrations:
0, 200 mg/kg/day (recovery group and 28 days females)
Basis:
actual ingested
No. of animals per sex per dose:: - Experimental group, F0: 10 (which resulted in 8 pregnant females)
- Recovery group and 28 days females: 5
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: based on the results of a 10-day dose range-finding study. In this study, TMP was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300, and 1000 mg/kg bw/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg bw/day and females at 300 and 1000 mg/kg bw/day. In addition, weight gain in males at 300 mg/kg bw/day was reduced by 43% compared to the controls. Therefore 300 mg/kg bw/day was considered too toxic for the OECD TG 422 study.
- Rationale for selecting satellite groups: were used as recovery animals to evaluate recovery from any possible treatment-related effects identified in the high-dose group
- Post-exposure recovery period in satellite groups: 2 weeks
Parental animals: Observations and examinations:: - CAGE SIDE OBSERVATIONS: Observations for mortality were made twice daily, and the general condition of all animals was checked daily.

- DETAILED CLINICAL OBSERVATIONS: Clinical examinations were conducted and recorded daily throughout the course of the study.

- BODY WEIGHT: The body weights of the F0 male rats were determined and recorded initially and then weekly until termination. Body weights of the 28-day females and recovery males and females were recorded on a weekly basis until termination. The body weights of F0 female rats were recorded in the same manner until conformation of mating. During gestation, F0 females were weighed on gestational days (GD) 0, 7, 14, and 20. Dams producing litters were weighed on post natal day (pnd) 0, 4, 7, 14, and 21. Any female that did not show evidence of successful mating after 14 days of cohabitation was continued on the original weekly weighing schedule.

- FOOD CONSUMPTION: Feed consumption was recorded weekly for all F0 parental animals during the 2-week prebreed exposure period and recorded weekly for the 28-day females. During pregnancy of the F0 females, feed consumption was measured for GD 0-7, 4-7, 7-14, and 14-21 (after pnd 14 this was confounded by the pups). Any female that did not show evidence of successful mating after 14 days of cohabitation continued on the original weekly schedule.

- HAEMATOLOGY: Blood was collected, from the tail vein, from 5 randomly selected F0 females per group on the last day of the prebreed exposure period (sd 13). Blood was collected prior to necropsy (sd 28) from 5 randomly selected F0 males per group and from the 28-day females. The parameters measured included evaluation of haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, RBC indices, and prothrombin time (PT), a measure of blood clotting time/potential (Becton Dickinson Fibrometer).

- CLINICAL CHEMISTRY: Blood was collected, from the tail vein, at necropsy (sd 28) from 5 randomly selected F0 males per group and from the 28-day females. Evaluations in serum included sodium, potassium, chloride, glucose, total cholesterol, blood urea nitrogen (BUN), creatinine, total protein and albumin, and 2 enzymes indicative for hepatocellular effects (alanine aminotransferase and aspartate aminotransferase)

- URINALYSIS: Prior to necropsy, 5 F0 males per group, randomly selected, and the 28-day females were singly housed overnight in metabolism cages. The total amount of time the animal was in the chamber and the amount of urine collected was recorded. The urine was evaluated for appearance and by dipstick analysis for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes.

- NEUROBEHAVIOURAL EXAMINATION: A functional observational battery (FOB), including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations, was performed on F0 males and 28-day females prior to dosing and weekly to termination at study day (sd) 28. A FOB was also performed on F0 females prior to dosing and weekly during prebreed, mating, gestation, and lactation and on recovery animals (male and female) prior to dosing, weekly during dosing, and once during the recovery period.
Litter observations:: PARAMETERS EXAMINED PREWEANING PERIOD: All F1 pups were individually counted, sexed, weighed, and examined grossly at birth (pnd 0), at pnd 4, 7, and 14, and at weaning (pnd 21). Anogenital distance was recorded with the individual pup weight on pnd 0. The presence or absence of retained nipples and areolae on the ventrum was recorded for F1 offspring males at pnd 11-13. All pups were examined for physical abnormalities (external developmental malformations) at birth and throughout the preweaning period. Survival indices were calculated on pnd 0, 4, 7, and 14 and at weaning.

PARAMETERS EXAMINED POSTWEANING PERIOD: F1 postweaning observations and procedures for each retained female included examination for VP (from pnd 22 until acquisition of vaginal opening) and determination of estrous cyclicity and normality, evaluated by vaginal smears taken daily the last 3 weeks of the postwean exposure period prior to the scheduled sacrifice. For each retained male offspring, observations for PPS began at 35 days of age and continued until acquisition of PPS. Body weights were recorded on the day of VP and PPS. All retained F1 weanlings were weighed and feed consumption measured once per week until their scheduled demise. Daily F1 mortality and clinical observations were conducted for the F1 animals during the last 3 weeks of the postwean exposure period. FOBs were performed on 5 randomly selected postwean F1 offspring/sex/group once, midway through the postwean period. Grip strength was determined in the last week of the postwean period for the same 5 animals/sex/group. In addition, blood was collected, and hematology, clinical chemistry (f1 males and females), and urinalysis (F1 males only) conducted on 5 F1 animals/sex/group prior to necropsy.

STANDARDISATION OF LITTERS: On pnd 4, the size of each litter was adjusted to 10 by eliminated extra pups by random selection to yield, as nearly possible, 5 males and 5 females per litter. Pups culled to standardize litters were euthanized and subjected to a complete gross necropsy.

GROSS EXAMINATION OF DEAD PUPS: Any F1 pup that appeared moribund or that died during lactation was necropsied, when possible, to investigate the cause of death and to identify internal visceral developmental malformations.

F1 MALE ANDROLOGY: At the time of sacrifice 1 testis from each F1 adult male was frozen at 20°C for subsequent enumeration of testicular homogenization-resistant spermatid heads for high-dose and control males. If treatment-related changes in the number of testicular homogenization resitant- spermatid heads were observed in the high-dose group, then these evaluations were extended to the mid-and low-dose group animals (from retained frozen testes). In addition, 1 cauda epididymis from each F1 male was immediately removed, weighed, and seminal fluid from the cauda assessed for sperm number, motility, and morphology. If treatment-related andrological changes were observed in the high-dose group, then these evaluations were extended to the mid- and then to the low-dose group animals.
Postmortem examinations (parental animals):: - GROSS PATHOLOGY: All F0 parental animals, 28-day females, recovery males and females were subjected to a complete gross necropsy, with selected organs weighted and retained or discarded as appropriate. The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities an their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin. Uretri of F0 females were examined for the number of nidation (implantation) scars.
- Organs weighed and retained for F0 animals, 28-day females and recovery animals: liver, kidneys, adrenals, thymus, heart, brain, spleen, testes, epididymides, prostate, seminal vesicles, ovaries, uterus with cervix + vagina.
- Organs only retained for F0 animals, 28-day females and recovery animals: spinal cord, thyroid, sciatic nerve, stomach, small/large intestine, trachea/lungs, urinary bladder, bone marrow (femur), lymph nodes, gross lesions.

- HISTOPATHOLOGY: Full histopathology was performed on all retained organs from 5 randomly selected F0 males and females in the control and high-dose groups as well as for the 28-day females.
Postmortem examinations (offspring):: - GROSS PATHOLOGY: The retained F1 adults were subjected to a complete gross necropsy, with selected organs weighted and retained or discarded as appropriate. The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities an their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin. All nonselected F1 weanlings were subjected to a complete gross necropsy. The retained F1 offspring were sacrificed at ca. 70 days of age and subjected to the same assessments as the F0 parents (expect for the nidation scars).
- Organs weighed and retained for F1 adult animals: liver, kidneys, adrenals, thymus, heart, brain, spleen, testes, epididymides, prostate, seminal vesicles, ovaries, uterus with cervix + vagina.
- Organs only retained for F1 adult animals: spinal cord, thyroid, sciatic nerve, stomach, small/large intestine, trachea/lungs, urinary bladder, bone marrow (femur), lymph nodes, gross lesions.
- Organs only weighed F1 weanlings: thymus, brain, spleen.
- Organs only retained F1 weanlings: gross lesions.
- Organs weighed and retained F1 weanlings: testes, epididymis, ovaries, uterus with cervix + vagina.

- HISTOPATHOLOGY: Full histopathology was performed on all retained organs from 5 randomly selected F1 males and females.
Statistics:: See any other information on materials and methods incl. tables
Reproductive indices:: The following indices were determined:
- Female mating index (%) = (number of females sperm positive/ number of females paired) x 100
- Female fertility index (%) = (number of females pregnant / number of females sperm positive) x 100
- Gestation index (%) = (number of females with live litters / number of females pregnant) x 100
- Male mating index (%) = (number of males impregnating females / number of males paired) x 100
- Male fertility index (%) = (number of males siring litters / number of males impregnating females) x 100
- Pregnancy index (%) = (number of pregnant females / number of males impregnating females) x 100
Offspring viability indices:: The following indices were determined:
- Live birth index (%) = (number of live pups at birth / total number of pups born) x 100
- 4-Day survival index (%) = (number of pups surviving 4 days (precull) / total number of live pups at birth) x 100
- 7-Day survival index (%) = (number of pups surviving 7 days / total number of live pups at 4 days (postcull)) x 100
- 14-Day survival index (%) = (number of pups surviving 14 days / total number of live pups at 7 days) x 100
- 21-Day survival index (%) = (number of pups surviving 21 days / total number of live pups at 14 days) x 100
- Lactation index (%) = (number of pups surviving 21 days / total number of lie pups at 4 days (postcull)) x 100
Dose descriptor:: NOAEL
Remarks:: General toxicity
Effect level:: 200 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: Highest dose tested
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: 200 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: Highest dose tested
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: 200 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: Highest dose tested
Reproductive effects observed:: not specified
Conclusions:: In the absence of any parental or offspring toxicity, the F0 male and female (either pregnant or nonpregnant) systemic no observable adverse effect level (NOAEL) was at least 200 mg/kg/day. The NOAEL for F0 reproductive toxicity was at least 200 mg/kg/day. The NOAEL for F1 offspring toxicity was also at least 200 mg/kg/day.
Executive summary:: In a GLP compliant combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, performed according to OECD 422, male and female CD (Sprague-Dawley [SD]) F0 rats were administered 2,4,6-Trimethyl Phenol (TMP, Mesitol) orally by gavage. These animals received 0, 10, 100, and 200 mg/kg/day at a dose volume of 5 ml/kg/day in Mazola® corn oil, 10 animals/sex/dose, for 2 weeks of prebreed exposure (males and females), 2 weeks of mating (males and females), and 3 weeks of gestation and lactation each (F0 females) for F0 parents, and direct dosing of selected F1 offspring from weaning through scheduled sacrifice, at least 7 weeks postweaning. Five additional F0 males per group from the control and 200 mg/kg/day groups were designated as recovery animals and held without dosing for 2 weeks after the F0 male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high-dose group. Five additional females each from the 0 and 200 mg/kg/day groups (designated "28-day females") were not mated and were terminated after 28 days of dosing. Similarly, 5 females each from the 0 and 200 mg/kg/day groups (designated "28-day recovery females") were dosed for 28 days and held without dosing for an additional 2 weeks as for the recovery group of males. The F0 males and the 28-day females were, therefore, tested in a protocol consistent with OECD Guideline No. 407. Body weights, feed consumption and clinical signs were recorded. A function observational battery observation was performed on all initial animals once during quarantine and at least once per week for F0 animals during prebreed, mating (both sexes), gestation, and lactation (F0 females) treatment periods and on 5 F1 females and 5 F1 males once midway during the postwean exposure period. After the 2-week prebreed exposure period, animals were randomly mated within treatment groups for a 2-week mating period to produce the F1 generation, with continuing exposure. All F0 parental animals, nonselected F1 weanlings, and retained F1 adults were necropsied with complete histologic evaluation of the 28-day females and for 5 selected F0 and F1 males and females in the 0 and 200 mg/kg/day groups. On the day of birth (postnatal day [pnd] 0), anogenital distance was measured and body weights recorded for all live F1 pups in all litters. F1 litters were culled on pnd 4 to yield, as nearly as possible, 5 males and 5 females per litter. The culled F1 pups were weighed, euthanized, and necropsied with complete external and visceral examinations. For the remaining F1 pups, survival indices were calculated at least weekly through weaning (pnd 21). At weaning, at least 1 female and 1 male (whenever possible) from each F1 litter were randomly selected for a total of 10/sex/group to continue treatment for 7 more weeks, with dosing for F1 selected pups begun on pnd 22 until all pups were at least 70 days of age. F1 postweaning observations and procedures for each retained F1 female included examination for vaginal patency (VP; from pnd 22 until acquisition of vaginal opening) and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last 3 weeks of the postwean exposure period prior to scheduled sacrifice. For each retained F1 male offspring, observations for cleavage of the balanoprepreputial gland (preputial separation; PPS) began at 35 days of age and continued until acquisition of PPS. Andrologic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, clinical biochemistry, and urinalysis (28-day females and males only) assays were performed at necropsy for all 28-day females, for 5 randomly selected parental F0 males and females per dose group, and for 5 F1 adult males and females per dose group. There were no indications of toxicity in the recovery males, 28-day females, or recovery females. There were no treatment related effects on reproductive parameters. There was no F0 offspring toxicity during lactation in either sex. Acquisition of puberty in F1 males and females was unaffected, and F1 postweanlings exhibited no systemic toxicity in either sex at any dose. In conclusion, in the absence of any parental or offspring toxicity, the F0 male and female (either pregnant or nonpregnant) systemic no observable adverse effect level (NOAEL) was at least 200 mg/kg/day. The NOAEL for F0 reproductive toxicity was at least 200 mg/kg/day. The NOAEL for F1 offspring toxicity was also at least 200 mg/kg/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 22,1996

Deviations:

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

-Sources: Charles River Laboratories (Atsugi production plant)
-Age at study initiation : 9 weeks
-Weight at study initiation : It was confirmed that the body weight range of the animal is average body weight ±20%. Females 190-235 g, males: 293-338 g
-Animals were uniquely identified and kept in their cages for five days prior to the start of the study to allow for acclimatisation to the laboratory conditions
1 male and female animal was considered for mating period per 1 cage
-Feeding : Solid feed for the test animal, which is sterilized by radiation sterilization (CRF-1, Oriental Yeast Company Ltd.). Administration Oral (Compulsory oral administration). The rat was administered once a day by using a disposable cylinder in which a gastric tube was set. The administration solution was added while stirring with a stirrer.
-Water : drinking water which has undergone ultraviolet radiation after filtration through 5 μm filter
Temperature acceptable range 19-25 °C
Relative humidity 48 – 62.3%
Lighting hours: 12 hours/day (7.00 – 19.00)

Route of administration:

oral: gavage

Vehicle:

other: 0.1w/v% Tween 80 + 0.5w/v% Sodium carboxymethylcellulose solution

Details on mating procedure:

- M/F ratio per cage: 1:1

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males : Total of 42 days (from before 14 days of mating till the day after completion of mating)
Females : 14 days before mating, during mating, pregnancy and up to lactation day 4
Females (satellite), 42 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 10, 60, 300 mg/kg bw/day
Basis:
other: Amount of solution administered: By considering 10 mL/kg, the fluid volume in each animal was calculated on the basis of body weight measured on the day of arrival

No. of animals per sex per dose:

Males, 12 (5 for recovery) ; females, 12; satellite females, 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The results of “Repeated administration toxicity test (Test number: B051509) for 14 days by oral administration using rats administered with 2, 4, 6-trimethylphenol” (dosage: 100, 300, 600 and 1000 mg/kg) conducted in the testing facility were set as the reference. Considering the results and the administration period of this test, a dosage of 300 mg/kg clearly forecasted the appearance of toxicity. Hereafter, the dosage was 60 mg/kg while decreasing approximately by a geometric ratio of 5 and the low dosage was set to 3 dosages of 10 mg/kg. Moreover, the control group for which only the medium (0.1w/v% Tween80 addition of 0.5w/v%CMC-Na) was administered was set.

Positive control:

not used

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes (see Appendix Study B051477)
- Time schedule and parameters checked:

GENERAL STATE: The administration period was observed twice a day (before administration and after administration). Other periods were observed once in the morning in 1 day.

BEHAVIORAL EXAMINATION: Observation of detailed symptoms (in home cage, at the time of handling and in open field) was carried out once before starting the administration, once a week during the administration period, and at any time after 13:00 till the sixth week (Pregnancy day and childbirth day of the female were not considered). The function test (Reactiveness to stimulation, Grip measurement) and the measurement of amount of motor activities were carried out after 13:00 of the sixth week. For 5 female animals of each group the measurement was carried out once when the birth date was near, and once during the breastfeeding period.

BODY WEIGHT: Males - on day 1, 8, 15, 22, 29, 36, 42, and 43. In addition, the weight of male recovery animals was measured on day 50 and 56. The weight of female satellite animals was measured with the same frequency as that of the male recovery animals. The weight of female test animal was measured on day 1, 8, and 15. The weight of pregnant female after mating was measured on day 0, 7, 14, and 20 and the weight of female during breast‐feeding after delivery was measured on day 0 and 4.

FOOD INTAKE : In the male test animal and the male recovery animal was measured on days 1~8, 8~15, 22~29, 29~36, 36~38, 43~50 and 52~56. The food intake in female satellite animals was measured on days 1~8, 8~15, 15~22, 22~29, 29~36, 36~42, 43~50 and 50~56. The food intake in the female test animal was measured with the same frequency as that of measurement of body weight. However, the food intake was not measured during the mating period when the animals were living together.

HAEMATOLOGY:
- Time schedule for collection of blood: day 42 in male test animal, day 56 in male recovery animals and female satellite animals, day 4 of lactation for female test animals).
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: yes (more than 16 hours)
- How many animals: 10 animals (5 males -5 females) per group
- Parameters checked: Red blood cell count - Hemoglobin concentration - Hematocrit value - Mean corpuscular volume (MCV) - Mean corpuscular hemoglobin (MCH) - Mean corpuscular hemoglobin concentration (MCHC) - Reticulocyte count - Blood platelet count - Prothrombin time (PT) - Activated partial thromboplastin time (APTT) - White blood cell count - Differential leukocyte count.

BIOCHEMICAL EXAMINATION:
OF BLOOD - Parameters checked: ASAT (GOT) - ALAT (GPT) – γGT – ALP - Total bilirubin - Urea nitrogen – Creatinine – Glucose - Total cholesterol – Triglyceride - Total protein – Albumin - A/G ratio – Calcium – Inorganic phosphorous - Sodium (Na) - Potassium (K) - Chloride (Cl)
OF URINE Parameters checked: ( test on 5 male once on the 38th day) pH - Protein - Glucose - Ketone body - Bilirubin - Occult blood - Urobilinogen

Oestrous cyclicity (parental animals):

In the morning from the day of starting the administration to the day of starting the hybridization, vaginal smear was collected; estrous cycle was examined and the incidence rate of average sexual cycle days and abnormal sexual cycle animals (animals whose sexual cycle is not 4~6 days) was calculated. Hybridization, observation of delivery and lactation and inspection after completion of breastfeeding were performed.

Litter observations:

PARAMETERS EXAMINED
The number of births (number of alive infants, number of stillborn infants), gender and presence of external abnormality, were examined on day 0 of lactation. Afterwards, general state and presence of death were observed every day. The weight was measured on 0 day and after 4 days of birth. External appearance that contains mouth cavity of the living infants was examined after 4 days of the birth.

Postmortem examinations (parental animals):

SACRIFICE:
Males, day 43 of treatment and day 15 of recovery.
Females, day 5 of lactation;
Females (satellite), day 15 of recovery

GROSS NECROPSY
- yes
Weight of below-mentioned organs were measured for 5 male test animals, for all the male recovery and the female satellite animals, and 5 female animal having early birth date: Brain, heart, liver, kidney, adrenal gland, thymus gland, spleen, testicles, epididymides

HISTOPATHOLOGICAL EXAMINATION
- Yes
Brain, pituitary gland, thymus gland, lymph node (lower jaw/mesentery), trachea, lung, stomach, intestinal tract (duodenum, jejunum, ileum, appendix, colon and rectum), thyroid gland, parathyroid gland (both sides), heart, liver, spleen, kidney (both sides), adrenal glands (both sides), urinary bladder, testicles (both sides), epididymides (both sides), seminal vesicles (including the coagulating gland), ventral prostate, ovaries (both sides), uterus, vagina, bone marrow (femur (one side)), sciatic nerve (one side), spinal cord, and abnormal parts that are visible by the naked eye.
For all abnormal parts visible with naked eyes, microscopic examination was carried out.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age. A dissection was performed after the euthanasia and a search for abnormal finding was performed

Statistics:

For the measurement data statistical significance Bartlett's test was used. When the variance was equal and when neither one-way analysis of the variance nor the variance was equal, the Kruskal-Wallis Test was conducted. When significant difference was observed between the groups the Dunnett methods or the Dunnett types was compared in multiple numbers. For some items, the Kruskal-Wallis Test was conducted. When significant difference was observed between the groups, the Dunnett types were compared in multiple numbers. Moreover, in histopathological examination, the enumeration data of 2 groups was compared with control group in the Wilcoxon rank sum test. Other enumeration data was tested by the Fisher's exact probability method. The data between 2 groups (control group and 300 mg/kg group) was analyzed conducting the Student's or the Welch's t-test. The enumeration data of 2 groups was compared with the control group by Wilcoxon rank sum test. The significance level of every test was assumed to be 5% using the safety test system MiTOX.
Multiple comparison tests:Weight, amount of food intake, hematological test, biochemical examination of blood, weight of organs, behavior examination measurement data (Strength of grip and motor activities), number of corpora lutea, number of implantations, number of born infants (number of alive infants, number of stillborn infants).
Multiple comparisons between Kruskal-Wallis type and Dunnett type: Urine test, Mating location and number of days, frequency of mating seasons missed till mating, average number of cycles, gestation period, implantation index, delivery rate, birth rate, incidence rate of external abnormality, viability index on day 4 of the newborn.
Wilcoxon rank sum test: Histopathological examination.
Fisher’s exact probability method: Incidence rate of abnormal estrous cycle, copulation rate, fertility index, birth rate, sex ratio.
F-test and Student’s t-test or Welch’s t-test: Variables data of recovered animal

Reproductive indices:

yes

Offspring viability indices:

yes (Appendix 39 Study B051477)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

M: Edema in the forestomach (300), Globule leukocyte in the glandular stomach ↑ (300); F: Erosion in the glandular stomach (300); M/F: Foveola hyperplasia in the glandular stomach (300), Squamous hyperplasia in the forestomach (60, 300).

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Hyperplasia of stomach squamous epithelium was noticed in both the male and the female groups of 60 mg/kg and above. Fertility was not affected.
No change in the test substance was noticed in case of the estrous cycle, copulation rate, conception rate, delivering rate, gestation period, corpus luteum, implantation, implantation rate, birth rate, delivery and breastfeeding behavior in the parent animals. For details on stomach epithelial hyperplasia it is referred to the chapter of repeated dose toxicity.

Dose descriptor:

NOEL

Remarks:

reproductive toxicity

Effect level:

300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: In reproductive toxicity,regarding NOEL as well as NOAEL in parent animal of male and female, no change was observed. As a result, 300 mg/kg/day dose was considered.

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

In case of the newborn baby, no change was noticed in the number of babies born, birth rate, gender ratio, survival rate within 4 days of the newborn baby, external appearance, general state, body weight, and dissection. In the dissection of living infants after 4 days of birth as well as in the dissection of dead infants, no abnormal findings noticed resulting from the test substance were noticed.

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Generation:

Effect level:

300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: In developmental toxicity, regarding NOEL as well as NOAEL in F1 baby animal of male and female, no change was observed. As a result, 300 mg/kg/day dose was considered.

Reproductive effects observed:

not specified

NOEL and NOAEL for reproduction toxicity on male and female parent animals and F1 baby animals were considered to be 300 mg/kg/day because no change was noticed.

Conclusions:

It was concluded from the above results that NOEL and NOAEL for the repeated dose toxicity of 2,4,6-Trimethylphenol were 10 mg/kg/day for both sexes, and that the NOEL and NOAEL for the reproductive/developmental toxicity were 300 mg/kg/day for parental animals and offspring.

Reproductive and developmental toxicity
No change resulting from the test substance was observed in estrous cycle, mating index, fertility index, delivery rate, pregnancy period, number of corpora lutea, number of implantations, implantation index, birth rate, and childbirth conditions and lactation.
In the examination of newborn baby, no change was observed in number of offsprings, number of alive infants, sex ratio, birth rate, and viability index on day 4 day of the newborn animal. Further, no change resulting from the test substance was observed in general state, external examination, weight, and dissection. Therefore, it is thought that the test substance does not exert influence on occurrence and development of the next generation.

Executive summary:

Repeated dose toxicity

2,4,6-Trimethylphenol was studied for oral toxicity in rats in accordance with the OECD guideline on combined repeated dose and reproductive/developmental toxicity screening test at dosages of 0, 10, 60, and 300 mg/kg/day.

No deaths occurred in any of the treated groups. In the 300 mg/kg group, salivation in both sexes and abnormal gait in females were noted as post-dose symptoms. Histopathological examination revealed squamous hyperplasia in the forestomach in the 60 and 300 mg/kg groups of both sexes, and edema in the forestomach in one male were noted.

In addition, foveola hyperplasia and erosion in the glandular stomach in the 300 mg/kg group were noted. Almost all the changes disappeared after a 14-day recovery period.

Reproductive and developmental toxicity

The compound had no effects on reproductive parameters such as the estrous cycle, mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition, or maternal behavior. On the

examination of neonates, there were no significant differences in the number of offspring or live offspring, sex ratio, live birth index, or viability index on day 4. No abnormal findings ascribable to the compound were found in the external features, clinical signs, body weights or necropsy of the offspring.

Conclusion

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 300 mg/kg bw/day
Study duration:: subacute
Experimental exposure time per week (hours/week):: 7
Species:: rat
Quality of whole database:: RL1. Information meets the requirements under REACH

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 a GLP compliant combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, performed according to OECD TG 422, male and female CD (Sprague-Dawley [SD]) F0 rats were administered the test substance orally by gavage (RTI International, 2005). These animals received 0, 10, 100, and 200 mg/kg bw/day at a dose volume of 5 mL/kg bw/day in corn oil, 10 animals/sex/dose, for 2 weeks of prebreed exposure (males and females), 2 weeks of mating (males and females), and 3 weeks of gestation and lactation each (F0 females) for F0 parents, and direct dosing of selected F1 offspring from weaning through scheduled sacrifice, at least 7 weeks postweaning. Five additional F0 males per group from the control and 200 mg/kg bw/day groups were designated as recovery animals and held without dosing for 2 weeks after the F0 male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high-dose group. Five additional females each from the 0 and 200 mg/kg bw/day groups (designated "28-day females") were not mated and were terminated after 28 days of dosing. Similarly, 5 females each from the 0 and 200 mg/kg bw/day groups (designated "28-day recovery females") were dosed for 28 days and held without dosing for an additional 2 weeks as for the recovery group of males. The F0 males and the 28-day females were, therefore, tested in a protocol consistent with OECD TG 407. Body weights, feed consumption and clinical signs were recorded. A function observational battery observation was performed on all initial animals once during quarantine and at least once per week for F0 animals during prebreed, mating (both sexes), gestation, and lactation (F0 females) treatment periods and on 5 F1 females and 5 F1 males once midway during the postwean exposure period. After the 2-week prebreed exposure period, animals were randomly mated within treatment groups for a 2-week mating period to produce the F1 generation, with continuing exposure. All F0 parental animals, nonselected F1 weanlings, and retained F1 adults were necropsied with complete histologic evaluation of the 28-day females and for 5 selected F0 and F1 males and females in the 0 and 200 mg/kg/day groups. On the day of birth (postnatal day [PND] 0), anogenital distance was measured and body weights recorded for all live F1 pups in all litters. F1 litters were culled on pnd 4 to yield, as nearly as possible, 5 males and 5 females per litter. The culled F1 pups were weighed, euthanized, and necropsied with complete external and visceral examinations. For the remaining F1 pups, survival indices were calculated at least weekly through weaning (PND 21). At weaning, at least 1 female and 1 male (whenever possible) from each F1 litter were randomly selected for a total of 10/sex/group to continue treatment for 7 more weeks, with dosing for F1 selected pups begun on pnd 22 until all pups were at least 70 days of age. F1 postweaning observations and procedures for each retained F1 female included examination for vaginal patency (VP; from PND 22 until acquisition of vaginal opening) and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last 3 weeks of the postwean exposure period prior to scheduled sacrifice. For each retained F1 male offspring, observations for cleavage of the balanoprepreputial gland (preputial separation; PPS) began at 35 days of age and continued until acquisition of PPS. Andrologic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, clinical biochemistry, and urinalysis (28-day females and males only) assays were performed at necropsy for all 28-day females, for 5 randomly selected parental F0 males and females per dose group, and for 5 F1 adult males and females per dose group. There were no indications of toxicity in the recovery males, 28-day females, or recovery females. There were no treatment related effects on reproductive parameters. There was no F0 offspring toxicity during lactation in either sex. Acquisition of puberty in F1 males and females was unaffected, and F1 postweanlings exhibited no systemic toxicity in either sex at any dose. In conclusion, in the absence of any parental or offspring toxicity, the F0 male and female (either pregnant or nonpregnant) systemic no observable adverse effect level (NOAEL) was at least 200 mg/kg bw/day. The NOAEL for F0 reproductive toxicity was at least 200 mg/kg bw/day. The NOAEL for F1 offspring toxicity was also at least 200 mg/kg bw/day.

In a GLP compliant combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, performed according to OECD TG422, male and female CD (Sprague-Dawley) F0 rats were administered the test substance at dosages of 0, 10, 60, and 300 mg/kg bw/day (Mitsubishi, 2007). No deaths occurred in any of the treated groups. In the 300 mg/kg group, salivation in both sexes and abnormal gait in females were noted as post-dose symptoms. Histopathological examination revealed squamous hyperplasia in the forestomach in the 60 and 300 mg/kg bw groups of both sexes, and edema in the forestomach in one male were noted. In addition, foveola hyperplasia and erosion in the glandular stomach in the 300 mg/kg bw group were noted. Almost all the changes disappeared after a 14-day recovery period. No change resulting from the test substance was observed in estrous cycle, mating index, fertility index, delivery rate, pregnancy period, number of corpora lutea, number of implantations, implantation index, birth rate, and pup conditions and lactation. No change was observed in number of offspring, number of alive pups, sex ratio, birth rate, and viability index on day 4 day post partum. Further, no change resulting from the test substance was observed in general state, external examination, weight, and gross pathology of the pups. Therefore, it was concluded that the test substance does affect reproduction and development. It was concluded from the results that the NOEL and NOAEL for the repeated dose toxicity of 2,4,6-Trimethylphenol were 10 mg/kg bw/day for both sexes, and that the NOEL and NOAEL for the reproductive/developmental toxicity were 300 mg/kg bw/day for parental animals and offspring.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD TG 414), rat: NOAELmaternal = 100 mg/kg bw/day; NOAELdevelopmental = 100 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 12 Oct - 28 Nov 2021
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: Adopted in 2018
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS:Puschino, Moscow region, Russia (www.spf-animals.ru)
- Females: Nulliparous and non-pregnant
- Age: Approximately 12 weeks at the initiation of dose administration on post-mating day 5
- Weight at study initiation: 184 g ± 15 g, N = 98 g
- Housing: Solid bottom polycarbonate cages (Type IV, 598 х 380 х 200 mm (LxWxH), 1820 cm sq., Tecniplast s.p.a.) with bedding. Females were group housed until mating, cohabited with males during mating (M/F ratio: 1:1), pregnant females were individually housed until scheduled sacrifice on gestation Day 20
- Diet: Mmucedola 4RF21 (Mmucedola s.r.l., Milanese MI, Italy) ad libitum
- Water: Filtered tap water ad libitum
- Acclimation period: Approximately 9 weeks

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES
From: 12 Oct 2021 To: 28 Nov 2021
Route of administration:: oral: gavage
Vehicle:: corn oil
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
Test item formulations were prepared every four days as a suspension of the test item in corn oil. The required amount of the test item (weighed separately for each concentration) was mixed with a calculated volume of corn oil and stirred using a magnetic stirrer until homogenous. The total volume of each concentration was aliquoted to the required volumes of days of administration and stored in tightly closed glass jars at room temperature. For the control group, the required aliquots of corn oil were placed in labelled jars and stored at room temperature. A daily record of the usage of formulation was maintained based on weights.

VEHICLE
- Justification for use and choice of vehicle: Test item has limited solubility in water
- Concentration in vehicle: 5, 20, 80 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. : MKCN9742
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: The stability of the test item in the vehicle (corn oil) prepared at concentrations of 5 and 80
mg/mL was confirmed following storage for 4 days at room temperature (the temperature
range, 20 – 25 °C) during the method validation study (Study no. 797/21). Analysis of formulations for homogeneity and concentration during the dosing period was conducted in the test facility at the beginning, in the middle, and at the end of the in-life phase using a validated method.
For homogeneity analysis, quadruplicate samples were collected from the top, middle, and
bottom strata of each dosing formulation prepared during the study. For concentration
analysis, quadruplicate samples were collected from the middle stratum of each dosing
formulation (including the vehicle control group) prepared during the study. Samples
collected from the mean stratum for homogeneity analysis were used for this purpose.
Acceptance criteria for the formulations analysis are based on the test item in vehicle
composition as a suspension. The actual concentration of analyzed samples collected from
the mean stratum of formulations should be within the range of 85% - 115% of the target
concentration. The acceptance criteria for homogeneity are RSD<15% (for suspensions),
with the mean concentration within 85 -115% of the target concentration.
Details on mating procedure:: - Impregnation procedure: cohoused
- M/F ratio per cage: 1:2
- Length of cohabitation: Until mating. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of pregnancy
Duration of treatment / exposure:: Day 5 to 19 of gestation
Frequency of treatment:: Daily
Duration of test:: From start of mating to last necropsy on Day 20 of gestation
Dose / conc.:: 25 mg/kg bw/day
Dose / conc.:: 100 mg/kg bw/day
Dose / conc.:: 400 mg/kg bw/day
No. of animals per sex per dose:: 24 to 25 females with confirmed mating in control and low dose group. 25 females with confirmed mating in mid and high dose group.
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: Dosage levels were selected for the study based on available data from a previously conducted dose range finding study (796/21 of BTC/BTL IBCh RAS). No mortality and morbidity were observed in female SD rats on the test item (with a purity of 82 %) at the doses of 100 and 400 mg/kg bw/day during 14-day oral treatment. The 400 mg/kg bw/day dose caused in 1/4 female rats transient hypotonia and ataxia, inability to move with a temporary prone position, diarrhoea, and salivation. A dose-related increase in the mean liver and kidney absolute and relative weights were revealed.
- Rationale for animal assignment: Before the beginning of the treatment period, the females with confirmed mating were allocated to the experimental groups according to a stratification procedure so that the average body weight of each group did not statistically differ. Females with the same day of gestation were allocated to a different group.
- Time of day for (rat) dam blood sampling: At scheduled necropsy on Day 20 of gestation (10:00 - 13:00 hours). Animals were not fasted prior to blood collection.
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon at the same time, for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each female was observed for signs of toxicity approximately 15 - 60 min following dose administration. In addition, the presence of findings at the time of dose administration was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Day 0, 5, 8,11,14,17, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: A complete necropsy was conducted on all females at scheduled termination. Necropsy
included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.
- Sacrifice on Gestation Day 20 of gestation
- Organs examined: Thyroid gland, Uterus and ovaries
- Organs weighed: Liver, kidney, thyroid and brain
- Histopathology: Thyroid gland
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:: - Plasma: No
- Serum: Yes
- Volume collected: not indicated
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes
Statistics:: Continuous data variables (mean body weights and food consumption data) were analyzed by multi-factor analysis of variance (ANOVA-2), followed by the Duncan test, to determine inter-group differences. Former implantation sites, number of corpora lutea, implantation loss indices, hormones concentration values, organ weights were analyzed by parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (0.05), Dunnett's test is applied to the data to compare the treated groups to the control group. The t-test was used additionally to compare each dose group with the control value. The number of male and female fetuses per litter, AGD value, and mean value of affected fetuses per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine the intergroup difference, and the t-test was applied to the AGD value to compare each dose from the control value. The fetal body weight was analyzed by sex as well as for both sexes combined using a one-way analysis of variance (ANOVA) as described above, and the t-test was applied to compare each dose from the control value. Additionally, statistical analysis for fetal body weight was done using covariant analysis with litter size as a covariant.
Indices:: Females with total resorptions (N)
Females with all dead fetuses (N)
Females with live fetuses (N)
Corpora lutea (N per animal)
Implantation sites (N per animal)
Pre-implantation loss (N per animal, % of implantation sites):(Number of corpora lutea – Number of -implantation sites) / Number of corpora lutea
Fetuses (N per animal)
Live fetuses (N per animal, % of implantation sites)
Dead fetuses (N per animal, % of implantation sites)
Resorptions + Scars (N per animal, % of implantation sites)
Implantation scars (N per animal, % of implantation sites)
Resorptions – early (N per animal, % of implantation sites)
Resorptions – late (N per animal, % of implantation sites)
Post-implantation loss (N per animal, % of implantation sites): (Number of implantation sites – Number of live fetuses) / Number of implantation sites
Historical control data:: Historical control data are shown in appended Tables
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: The test item related external observations were noted in the 400 mg/kg bw/day dose group. Prone position was recorded for one female (No.70) following GD 13 - 15 dose administration. This clinical observation was temporary, observed approximately 30 - 40 min after dosing and was associated with hypersalivation on GD 13. The observation was considered adverse.
Dermal irritation (if dermal study):: not examined
Mortality:: no mortality observed
Description (incidence):: There was no morbidity and mortality of females caused by the test item administration.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Summary data of body weight and body weight gain for pregnant females are presented in Table 1 and 2 under 'Any other information an results incl. tables'.
A slight decrease in the mean body weight of pregnant females at 400 mg/kg bw/day (4.6 % compared to the control group) towards the end of the dosing period was not significant. However, body weight gain was statistically reduced in the high dose group starting from GD 17 compared to the control (6.5%). By GD 20, the body weight gain in the high dose group was decreased by 8.3 % compared to the control group.
However, the mean gravid uterus weight in the 400 mg/kg bw/day dose group was less than the control (17.4 % (p < 0.05)) and the maternal body weight without gravid uterus did not statistically differ from the body weight of control females. Therefore the treatment-related bodyweight effects were not considered to be adverse.
Food consumption and compound intake (if feeding study):: no effects observed
Description (incidence and severity):: Mean food consumption in the 25, 100, and 400 mg/kg bw/day treatment groups was similar to that of the vehicle control group during all study days.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Endocrine findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, non-treatment-related
Description (incidence and severity):: Females at 400 mg/kg bw/day had slightly increased relative kidney (+4.3%) and thyroid (+6.8%) weights. However, this change was not significant compared to the control group and were not considered to be biologically relevant.
Gross pathological findings:: effects observed, non-treatment-related
Description (incidence and severity):: No gross findings related to the test item administration were observed.

Spleen deformation was found in one female from the 100 mg/kg bw/day dose group.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: No histological alterations in the thyroid gland, which are considered to be associated with the test item treatment, were observed. The incidence of follicular cell hypertrophy was approximately the same among all groups (7/23, 7/24, 5/24, 5/23 in control, low, mid and high dose groups respectively). This finding was of slight or moderate grade in single females, including one control, not correlated to the change in thyroid weight and thyroid hormone levels, and so considered not test item related.
The incidence of ultimobranchial cyst was approximately the same among all groups (7/23, 7/24, 11/24, 9/23 in control, low, mid and high dose groups respectively). This finding was considered congenital, not treatment-related.
Histopathological findings: neoplastic:: not examined
Other effects:: effects observed, non-treatment-related
Description (incidence and severity):: There were no significant changes in the T3, T4, and TSH hormone levels in treated females compared to the control vehicle group.
The mean level of TSH in the 25 mg/kg bw dose group was lower compared to values in other groups; however, this change is considered incidental, not test item related. Considering that the level of T4 was unaffected, it was assumed that the TSH level did not change under the test item influence. There were no indications of TSH-mediated thyroid gland activation, such as a significant increase in thyroids weight.
Number of abortions:: no effects observed
Pre- and post-implantation loss:: effects observed, treatment-related
Description (incidence and severity):: The mean number of corpora lutea was approximately the same among groups. However, the mean number of implantation sites was decreased in the 400 mg/kg bw/day dose group (by 17.8% compared to control vehicle group, p < 0.05). Accordingly, the mean number of pre-implantation loss and percentage of pre-implantation loss per group were increased: 3.2 ± 3.4 and 1.5 ± 1.7 (p < 0.05) in animals of the high dose and control group, respectively and 26.8% and 12.1% (p < 0.01) in animals of the high dose and control group, respectively. The percentage of pre-implantation loss in the 400 mg/kg bw/day dose group was also statistically significantly higher than the historical control value of 16.7%. According to previous studies, implantation sites in rats can be visualized (using intravenously Evans blue) 5 days 12 h later mating [Novaro V. et al., 2002; Hamilton G. et al. 1994]. In this study, the test item administration started at this time (approximately 5.5 days after mating). Therefore, it is likely that the first administered dose of 400 mg/kg bw/day can adversely affect the implantation process.
Summary data are shown in Table 3 under 'Any other information an results incl. tables'.
Total litter losses by resorption:: no effects observed
Description (incidence and severity):: There were no total litter losses by resorption.
Early or late resorptions:: effects observed, non-treatment-related
Description (incidence and severity):: The mean number of fetuses per animal was reduced in the 400 mg/kg bw/day dose groups (by 18%, p < 0.05, compared to the control group). This change correlated to the decrease in the number of implantation sites in this group discussed above and assumed to be test item related.
The percentage and the mean number of early and late resorptions and total post-implantation loss were not statistically significantly changed compared to the control vehicle group. The number of females with post-implantation loss was approximately the same in all groups (13/23, 12/24, 8/24, 11/23 in control, low, mid and high dose group respectively. Thus, in the 400 mg/kg bw/day dose group, the decrease in the mean number of fetuses per uterus was associated with the pre-implantation loss. No increase in post-implantation loss was observed for the test item at doses of 25, 100 and 400 mg/kg bw/day.
Summary data are shown in Table 4 under 'Any other information an results incl. tables'.
Dead fetuses:: no effects observed
Description (incidence and severity):: No dead foetuses were observed
Changes in pregnancy duration:: no effects observed
Description (incidence and severity):: All pregnant females reached GD 20
Changes in number of pregnant:: no effects observed
Other effects:: not examined
: Key result
Dose descriptor:: NOAEL
Effect level:: 100 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: behaviour (functional findings)
Remarks on result:: other: One female in the 400 mg/kg bw/day dose group was prone following dosing on GD 13 - 15
Fetal body weight changes:: effects observed, treatment-related
Description (incidence and severity):: The mean body weight of male fetuses was slightly decreased (by 5.2 %) for doses of 100 and 400 mg/kg bw/day compared to the control vehicle group. This change correlated to the increased incidence of external observation of “small fetus” in these groups and was considered test item-related. However, the decrease in fetal body weight was not statistically significant and therefore considered non-adverse.
The test item treatment did not change the body weight of female fetuses and mean values for all fetuses.
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: All fetuses were found to be alive.
Changes in sex ratio:: effects observed, treatment-related
Description (incidence and severity):: The mean number of males per litter was statistically significantly reduced in the high dose group compared to the control vehicle group (p < 0.05). The percentage of males was decreased in this group (44.7%) compared to the value in the control group (51.9%). A decreased percentage of males per group was observed for the 25 and 100 mg/kg bw/day dose groups (46.4% and 46.9% respectively). The reduction in ratio of male and female fetuses was not statistically significant, without clear dose-response relationship and was within historical control range (53.0% for 70 litters; min–max per litter, 14–79%) and considered to be non-adverse.
Changes in litter size and weights:: effects observed, treatment-related
Description (incidence and severity):: The mean number of males per litter in the 400 mg/kg bw/day dose group was statistically reduced compared to the control (p < 0.05), and the percentage of males
was decreased in this group (44.7%) compared to the value in the control group (51.9%). A decrease in the percentage of males per group was observed at 25 and
100 mg/kg bw/day dose level (46.4% and 46.9% respectively). The reduction in ratio of male and female fetuses was not statistically significant, without clear dose relation and was within historical control range (53.0% for 70 litters; min–max per litter, 14–79%) and considered to be non-adverse. The mean body weight of male fetuses in the 100 and 400 mg/kg bw/day dose groups was slightly decreased compared to the control vehicle group. This change (5.2% in both groups) was not statistically significant and considered non-adverse. The test item treatment did not affect the body weight of female fetuses and mean values for all fetuses.
Anogenital distance of all rodent fetuses:: no effects observed
Description (incidence and severity):: The absolute and normalized anogenital distance in male and female fetuses was not
significantly changed in all dose groups.
Changes in postnatal survival:: not examined
External malformations:: effects observed, non-treatment-related
Description (incidence and severity):: The total affected fetuses were approximately the same among all groups.
In the test item treated groups, the following external malformations were revealed for single fetuses.
Umbilical hernia was observed in 3/231 (2 litters affected), 1/233. 0/243. 2/188 (2 litters affected) fetuses in the control, low, mid and high dose respectively. This observation has low incidence in the historical control population (2/878 fetuses, 2 litters affected) and was associated in the present study in most cases with generalized subcutaneous oedema (anasarca), which was observed in the approximately same incidence in all groups including control.
One fetus in the 100 mg/kg bw/day dose group had a malformed forepaw unilaterally. The absence of all carpals, metacarpals, and phalanges on this forelimb was shown. Although such a finding was not shown in the historical control population, this single not dose depended malformation was considered not test item related.
Cleft palate was found in a single fetus from the 25 mg/kg bw/day dose group. This fetus had generalized subcutaneous oedema, domed head, and umbilical hernia and was small in size. So, the cleft palate associated with complex findings in a single fetus in the low dose group was considered not test item related.
Skeletal malformations:: effects observed, treatment-related
Description (incidence and severity):: The increase in the fetal incidence of delayed ossification, which was considered to be caused by the test item, was revealed for some skeletal structures starting from 25 mg/kg bw/day.
In the 25 mg/kg bw/day dose group, the increase in fetal incidence of unossified forelimb phalanges (70.1% versus 46.5%, p < 0.001), unossified 7th cervical vertebra (70.9% versus 57.9%, p < 0.05), incomplete ossification of caudal vertebrae (30.8% vesus 14.9%, p < 0.01) was observed.
In the 100 mg/kg bw/day dose group, the increase in fetal incidence of unossified forelimb phalanges (65.6% versus 46.5%, p < 0.001), increase in fetal and litter incidence of unossified 5th sternebra (13.9% versus 2.6%, p < 0.01, and 41.7% versus 13.0%, p < 0.05), in fetal incidence of unossified 6th sternebra (18.0% versus 8.8%, p < 0.05) were revealed.
In the 400 mg/kg bw/day group, incomplete ossification of interparietal skull bone was observed with fetal incidence of 7.5% (versus 1.8%, p < 0.05) and litter incidence of 30.4% (versus 8.7%). The incidence of unossified 6th sternebra was increased to 18.3% compared to 8.8% in the control group (p < 0.05). The increase in the incidence of unossified forepaw phalanges and delayed ossification in the caudal vertebrae was approximately the same as in low and medium dose groups. Moreover, in the high dose group, a statistically significant increase in the fetal incidence of an unossified first thoracic vertebra is recorded (7.5% versus 1.8%, p < 0.05).
All findings of delayed ossification are considered skeletal variations to indicate fetal immaturity and a transient stage in fetal development correlated to the slight decrease in body weight and are not thought to be adverse.
Visceral malformations:: effects observed, treatment-related
Description (incidence and severity):: No malformations of soft tissues were found in any observed fetuses.
In the 100 and 400 mg/kg bw/day dose group, a slight increase in the fetal incidence of dilatation of brain ventricles was observed (9.1% and 10.5% compared to 4.2% in the control group). This finding was dose-dependent and frequently found in small fetuses (8 of 11 fetuses with ventricles alteration at 100 mg/kg bw/day, and 5 of 10 fetuses at 400 mg/kg bw/day dose) and considered to be test item related. However, the effect was not statistically significant and is not thought to be adverse.
Other visceral findings were observed in single cases without dose-dependence or with approximately the same fetal and litter incidence among all groups, including control, and so they are considered not treatment-related.
Other effects:: not examined
: Key result
Dose descriptor:: NOAEL
Effect level:: 100 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: reduction in number of live offspring
Remarks on result:: other: Decreased number of fetuses per litter at 400 mg/kg bw/day which correlated to increased pre-implantation loss.
: Key result
Developmental effects observed:: yes
Lowest effective dose / conc.:: 400 mg/kg bw/day
Treatment related:: yes
Relation to maternal toxicity:: developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:: no
Relevant for humans:: presumably yes
Conclusions:: Under the conditions of the present study the test substance did not induce any treatment-related biologically relevant malformations in the developing unborn organism in the presence of maternal toxicity. Therefore, the test substance does not meet the criteria for classification for prenatal developmental toxicity according to Regulation (EC) No 1272/2008. The available data is thus conclusive but not sufficient for classification.
Executive summary:: A prenatal developmental toxicity study was conducted to evaluate the potential effects of the test item, 2,4,6-trimethylphenol, on pregnancy and on embryo-fetal development in rats following daily oral (gavage) administration at doses 25, 100 and 400 mg/kg body weight (bw)/day from implantation to the day prior to scheduled caesarean section (Day 5 to Day 19 post-mating inclusive) (Khokhlova, 2022). The study was carried out in accordance to the OECD Guideline 414: Prenatal Developmental Toxicity Study (2018) in GLP conditions. The test item, 2,4,6-trimethylphenol, in the vehicle (corn oil), was administered by gavage once daily to Sprague-Dawley (SD) female rats from Day 5 to Day 19 post-mating (inclusive). Three animal groups were received the test item at dose levels of 25, 100 or 400 mg/kg bw/day. A concurrent vehicle control group has received the vehicle (corn oil) on a comparable regimen and in the same volume of 5 mL/kg bw. Each group consisted of 24 - 25 females with confirmed mating. All females were observed twice daily for mortality and morbidity and signs of toxicity following dose administration. Body weights and food consumption were recorded at three-day intervals. On post-mating Day 20, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora lutea. The selected organs were weighed, and the weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of serum concentration of thyroxin (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were conducted in dams to observe pathological changes in thyroid function. Gravid uteri including cervix were weighed, and uteri content was examined to record implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external examination. Approximately half of the fetuses from each litter were subjected to a detailed examination of soft tissue by serial sectioning after fixation in Bouin’s solution, while the other half underwent detailed skeletal examination following staining of bone with alizarin red and cartilage with alcian blue. The fetal findings were classified as malformations or variations. Fetal incidence, litter incidence, and affected fetuses per litter were calculated for external, visceral, and skeletal alterations. The reproductive tract was examined with particular attention, and external sex was compared with internal (gonadal) sex in all fetuses; any incomplete testicular descent was noted in male fetuses. No mortality was observed in treated or control dams. Temporary prone position was recorded for one female after each 400 mg/kg bw/day dose administration starting from Day 13; hypersalivation was recorded in this female on Day 13. These effects were considered to be treatment related and adverse. There were no treatment related effects on bodyweight or food consumption. There were no treatment related effects observed following gross necropsy or following histopathology of the thyroid glands. There were no significant changes in the T3, T4, and TSH hormones level in test-treated females compared to the control vehicle group. The mean number of implantation sites was decreased in the 400 mg/kg bw/day treated females (by 17.8% compared to control group, p < 0.05), and the pre-implantation loss (%) was statistically higher when compared to the control group (26.8% versus 12.1%, p < 0.01) and higher than the historical control value of 16.7%. The mean number of fetuses per uterus was reduced in the 400 mg/kg bw/day dose group (18%, p < 0.05, compared to control group) which correlated with the decrease in the number of implantation sites at this dose. This finding was considered to be adverse. The percentage and the mean number of early and late resorptions and total postimplantation loss were not statistically changed compared to the control vehicle group. The number of females with post-implantation loss was approximately the same in all groups. There were no post-implantation embryo/fetal loss. There were no fetuses with test item related external, visceral or skeletal malformations. The body weight of male fetuses were non-significantly decreased and were associated with the delayed ossification in interparietal skull bone, forelimb phalanges, 6th sternebrae, 1st thoracic and caudal vertebrae. Incidences of delayed ossification in these bones were increased starting from the 25 mg/kg bw/day dose correlating to the slight decrease in fetal body weight and with values that were within the historical control data range or slightly exceeding it. Effects indicated fetal immaturity and considered to be variations and therefore not adverse.
It was concluded from this study that the dosage of 100 mg/kg bw/day was the maternal no observed adverse effect level (NOAEL). Due to the reduced number of fetuses per uterus correlated to the increased pre-implantation loss on 400 mg/kg bw/day dose, the NOAEL for embryo-fetal survival and development is 100 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 100 mg/kg bw/day
Study duration:: subacute
Experimental exposure time per week (hours/week):: 7
Species:: rat
Quality of whole database:: RL 1 - The available information meets the information requirements under REACH

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available (further information necessary)

Additional information

A prenatal developmental toxicity study was conducted to evaluate the potential effects of the test substance, on pregnancy and on embryo-fetal development in rats following daily oral (gavage) administration at doses 25, 100 and 400 mg/kg body weight (bw)/day from implantation to the day prior to scheduled caesarean section (Day 5 to day 19 post-mating inclusive) (Khokhlova, 2022). The study was carried out in accordance to the OECD TG 414: Prenatal Developmental Toxicity Study (2018) in GLP conditions. The test substance, in the vehicle (corn oil), was administered by gavage once daily to Sprague-Dawley (SD) female rats from Day 5 to Day 19 post-mating (inclusive). Three animal groups were received the test item at dose levels of 25, 100 or 400 mg/kg bw/day. A concurrent vehicle control group has received the vehicle (corn oil) on a comparable regimen and in the same volume of 5 mL/kg bw. Each group consisted of 24-25 females with confirmed mating. All females were observed twice daily for mortality and morbidity and signs of toxicity following dose administration. Body weights and food consumption were recorded at three-day intervals. On post-mating Day 20, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora lutea. The selected organs were weighed, and the weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of serum concentration of thyroxin (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were conducted in dams to observe pathological changes in thyroid function. Gravid uteri including cervix were weighed, and uteri content was examined to record implantation sites, early and late resorptions, dead and live fetuses. The fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external examination. Approximately half of the fetuses from each litter were subjected to a detailed examination of soft tissue by serial sectioning after fixation in Bouin’s solution, while the other half underwent detailed skeletal examination following staining of bone with alizarin red and cartilage with alcian blue. The fetal findings were classified as malformations or variations. Fetal incidence, litter incidence, and affected fetuses per litter were calculated for external, visceral, and skeletal alterations. The reproductive tract was examined with particular attention, and external sex was compared with internal (gonadal) sex in all fetuses; any incomplete testicular descent was noted in male fetuses. No mortality was observed in treated or control dams. Temporary prone position was recorded for one female after each 400 mg/kg bw/day dose administration starting from Day 13; hypersalivation was recorded in this female on Day 13. These effects were considered to be treatment related and adverse. There were no treatment related effects on bodyweight or food consumption. There were no treatment related effects observed following gross necropsy or following histopathology of the thyroid glands. There were no significant changes in the T3, T4, and TSH hormones level in test-treated females compared to the control vehicle group. The mean number of implantation sites was decreased in the 400 mg/kg bw/day treated females (by 17.8% compared to control group, p < 0.05), and the pre-implantation loss (%) was statistically higher when compared to the control group (26.8% versus 12.1%, p < 0.01) and higher than the historical control value of 16.7%. The mean number of fetuses per uterus was reduced in the 400 mg/kg bw/day dose group (18%, p < 0.05, compared to control group) which correlated with the decrease in the number of implantation sites at this dose. This finding was considered to be adverse. The percentage and the mean number of early and late resorptions and total postimplantation loss were not statistically changed compared to the control vehicle group. The number of females with post-implantation loss was approximately the same in all groups. There were no post-implantation embryo/fetal loss. There were no fetuses with test item related external, visceral or skeletal malformations. The body weight of male fetuses were non-significantly decreased and were associated with the delayed ossification in interparietal skull bone, forelimb phalanges, 6th sternebrae, 1st thoracic and caudal vertebrae. Incidences of delayed ossification in these bones were increased starting from the 25 mg/kg bw/day dose correlating to the slight decrease in fetal body weight and with values that were within the historical control data range or slightly exceeding it. Effects indicated fetal immaturity and considered to be variations and therefore not adverse.
It was concluded from this study that the dosage of 100 mg/kg bw/day was the maternal no observed adverse effect level (NOAEL). Due to the reduced number of fetuses per uterus correlated to the increased pre-implantation loss on 400 mg/kg bw/day dose, the NOAEL for embryo-fetal survival and development is 100 mg/kg bw/day.

Justification for classification or non-classification

The available data on reproduction of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

GROUP:	Control	2 (25 mg/kg bw/day)	3 (100 mg/kg bw/day)	4 (400 mg/kg bw/day)
	MEAN ± SD	MEAN ± SD	MEAN ± SD	MEAN ± SD
	N=23	N=24	N=24	N=23
Gestation Day	Body weight, g
0	168 ± 15	167 ± 15	167 ± 15	168 ± 14
5	184 ± 16	184 ± 16	181 ± 16	186 ± 14
8	192 ± 16	192 ± 16	189 ± 17	193 ± 14
11	203 ± 15	203 ± 17	201 ± 16	204 ± 15
14	214 ± 17	214 ± 17	212 ± 16	214 ± 16
17	235 ± 18	231 ± 21	233 ± 18	226 ± 23
20	263 ± 20	258 ± 22	260 ± 21	251 ± 21
Period, gestation days	Body weight gain, %
5-8	4.4 ± 1.7	4.7 ± 2.4	4.6 ± 3.1	3.6 ± 1.8
5-11	10.8 ± 2.5	10.7 ± 2.6	11.0 ± 3.5	9.4 ± 2.5
5-14	16.8 ± 2.7	16.7 ± 2.8	17.5 ± 3.3	14.9 ± 2.8
5-17	28.0 ± 3.7	25.9 ± 6.7	28.8 ± 4.2	21.5 ± 9.4 (a)(c)(d)
5-20	43.4 ± 5.8	40.8 ± 8.1	43.7 ± 6.7	35.1 ± 8.3 (a)(b) (d)

GROUP:	Control	2 (25 mg/kg bw/day)	3 (100 mg/kg bw/day)	4 (400 mg/kg bw/day)
	MEAN ± SD	MEAN ± SD	MEAN ± SD	MEAN ± SD
	N=23	N=24	N=24	N=23
Body weight at necropsy on gestation day 20, g	263 ± 20	258 ± 22	259 ± 21	251 ± 20
Gravid uterine weight, g	54.6 ± 12.3	51.9 ± 12.8	53.3 ± 8.0	45.1 ± 18.5 (a)
Final body weight without uterus, g	208 ± 19	206 ± 19	205 ± 18	205 ± 25

	Historical Control	Control	25 mg/kg bw/day	100 mg/kg bw/day	400 mg/kg bw/day
	(N=70)	(N=23)	(N=24)	(N=24)	(N=23)
Number of corpora lutea
Total, N	1102	280	290	293	276
MEAN ± SD	15.7 ± 3.0	12.2 ± 1.6	12.1 ± 1.4	12.2 ± 1.8	12.0 ± 2.2
Number of implantation sites
Total, N	918	246	247	255	202
MEAN ± SD	13.1 ± 2.2	10.7 ± 1.9	10.3 ± 2.3	10.6 ± 1.2	8.8 ± 3.2 (b)
Pre-implantation loss
Total, N	184	34	43	38	74 (a)
% of corpora lutea	16.7 %	12.1 %	14.8 %	13.0 %	26.8 % (a)
MEAN ± SD, N	2.6 ± 2.6	1.5 ± 1.7	1.8 ± 2.2	1.6 ± 1.7	3.2 ± 3.4 (b)

		Historical Control	Control	25 mg/kg bw/day	100 mg/kg bw/day	400 mg/kg bw/day
Number of treated females with confirmed mating	N		24	24	25	25
Number of pregnant females	N_pregnant	70	23	24	24	23

Implantation sites	N	918	246	247	255	202
N per animal	MEAN ± SD	13.1 ± 2.2	10.7 ± 1.9	10.3 ± 2.3	10.6 ± 1.2	8.8 ± 3.2 (a)

Resorptions - Early	N	32	13	11	10	14
% of implantation sites		3.5	5.3	4.5	3.9	6.9
N per animal	MEAN ± SD	0.5 ± 0.7	0.6 ± 0.7	0.5 ± 0.5	0.4 ± 0.8	0.6 ± 0.7

Resorptions - Late	N	8	2	2	2	0
% of implantation sites		0.9	0.8	0.8	0.8	0.0
N per animal	MEAN ± SD	0.1 ± 0.4	0.1 ± 0.1	0.1 ± 0.3	0.1 ± 0.3	0.0 ± 0.0

Post-implantation Loss	Total	40	15	14	12	14
% of implantation sites		4.4	6.1	5.7	4.7	6.9
N per animal	MEAN ± SD	0.6 ± 1.0	0.7 ± 0.6	0.6 ± 0.7	0.5 ± 0.8	0.6 ± 0.7
Number of females with post-implantation loss	N / N _pregnant	28 / 70	13 / 23	12 / 24	8 / 24	11 / 23

Total Fetuses	N	878	231	233	243	188
Alive	%	100.0	100.0	100.0	100.0	100.0
Dead	%	0.0	0.0	0.0	0.0	0.0
No. per animal	MEAN ± SD	12.5 ± 2.3	10.0 ± 1.9	9.7 ± 2.4	10.1 ± 1.7	8.2 ± 3.3 (a)

Registration Dossier

Registration Dossier

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Diss Factsheets

2,4,6-trimethylphenol

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Reference

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all