Registration Dossier

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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed per OECD 422 and following GLP. A subset of the animals tested were evaluated to a protocol similar or equivalent to OECD 407 (28 day repeat dose oral - rodent). The reliability of this study for the substance tested is a K1, but in application of read-across to a different substance ECHA’s guidance specifies that the score can be a maximum of K2. A justification for read-across is provided.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
This study exceeded the std OECD 422 design by following the F1 offspring to adulthood, with continued exposure and assessments of neurologic, immunologic, and reproductive structures and functions. The protocol also assessed F0 recovery males and 28-day
GLP compliance:
yes
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: P = 63 days old
- Housing: singly in polycarbonate cages with bedding except during mating.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-77 degrees F
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light cycle

IN-LIFE DATES: From: 2003-09-2 To: 2004-01-06
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil

- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1 male to 1 female
- Length of cohabitation: 14 days
- Proof of pregnancy: Observation of a vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All dosing formulations were analyzed for concentration verification. Standards for acceptable accuracy of mixing were that the means of the analyzed samples was within +/- 10% of the nominal concentration and the relative standard deviation for triplicate samples did not exceed 10%.
Duration of treatment / exposure:
F0 females were dosed premating through the day prior to necropsy.
Recovery males, females and 28 days females were dosed for 28 days.
F1 animals were dosed post-weaning until the day before scheduled necropsy, at least 7 weeks duration.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 10, 100, and 200 mg/kg/day
Basis:
nominal conc.
at a dose volume of 5 mL/kg/day
No. of animals per sex per dose:
F0 generation: 10 males and 10 females plus 5 addition males and females each in the control and high dose groups designated recovery animals.

In addition, 10 females (5 control and 5 in the high dose group) designated as the 28 day females were dosed for 28 days and necropsied.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a dose range finding study, the test substance was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300, and 1000 mg/kg/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day. In addition, weight gain in males at 300 mg/kg/day was reduced by 43% compared to the controls. Therefore, 300 mg/kg/day was considered too toxic for the OECD 422 study design.
- Rationale for animal assignment (if not random): Randomization stratified by body weight
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table [No.?] were included. twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily and included changes to skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, somatomotor activity, and behavior patterns.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during prebreeding, for F0 females during gestation (gd 0,7,14, and 21) and lactation (pnd 0, 4, 7, 14, and 21). Body weights of the 28 day females and recovery males and females were recorded on a weekly basis until termination. Body weight gains were computed.

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


Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were weighed, euthanized, necropsied and completed external and visceral examinations performed.

PARAMETERS EXAMINED
The following parameters were examined in surviving F1 offspring: For the remaining F1 pups, survival indices were calculated at least weekly through weaning (pnd21).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

OTHER:
At weaning at least one male and one female from each litter (when possible) 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 retained F1 females included examination of vaginal patency and determination of estrous cyclicity and normality evaluated by vaginal smears taken daily the last three weeks of the postwean exposure period prior to scheduled sacrifice. For each retained male offspring, observationsfor cleavage of the balanopreputial gland began at day 35 and continued until preputial separation. Androgenic assessments were also performed on the F1 retained males at necropsy. In addition, hematology, and clinical chemistry were performed at necropsy for 5 randomly selected F1 adult male and females per dose group.

Body weights for selected F1 offspring from weaning through scheduled sacrifice were taken.

Functional Observation Battery (FOB), including cage side observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations was performed on 5 F1 females and 5 F1 males once midway during the postwean exposure period. Grip strength was also assessed for the 5 F1 males and 5 F1 females per group selected for FOB during the last week of the post weaning exposure.
Postmortem examinations (parental animals):
GROSS NECROPSY and HISTOPATHOLOGY
-All F0 parental animals, 28-day females, recovery males and females, adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of F0 females were examined for the number of nidation (implantation) scars. All nonselected F1 weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. A 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. Since no treatment-related findings were identified, no histopathology on lower dose groups or recovery groups was performed.
Postmortem examinations (offspring):
Retained F1 adults were subjected to a complete gross necropsy, with selected organs weighed and retained or discarded . The gross necropsy included examination of the external surfaces, all orifices, the carcass, the thoracic, abdominal, and pelvic cavities and their viscera, and cervical tissues and organs. Gross lesions were recorded and retained in 10% neutral buffered formalin, with selected organs weighed and retained or discarded, as appropriate. Uteri of F0 females were examined for the number of nidation (implantation) scars. All nonselected F1 weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. The retained F1 offspring were sacrificed at 70 days of age and subjected to the same assessments as the F0 parents.
Full histopathology was performed on all retained organs from 5 randomly selected F1 males and females in the control and high-dose groups. Since no treatment-related findings were identified, no histopathology on lower dose groups was performed.

Statistics:
Appropriate statistical analyses were used throughout the assay.
Reproductive indices:
At the time of sacrifice, 1 testis from each F1 adult male was frozen at -20 degrees 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-resistant 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. Sperm motility (motile and progressively motile) was assessed immediately after necropsy for all males. The number and morphology (at least 500 sperm per male, if possible) was evaluated at a later date using appropriately retained sperm samples initially from the high-dose and control males. 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 if treatment-related changes were seen in the mid-dose group (from retained sperm samples).

The mating index, fertility index and pregnancy index were all calculated for males and females.

F1 female age acquisition of vaginal patency was evaluated and the estrous cycle length monitored for the last 3 weeks of the post wean period for the F1 females.
Offspring viability indices:
For the offspring, the live birth index, the 4, 7, 14 and 21 day survival index, as well as the lactation index were all calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
rooting post dosing -consistent with taste aversion
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:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
F0 Reproductive Indices
There were no significant effects of exposure to the test substance on F0 mating, fertility, gestational, or pregnancy indices in F0 parental males and females during the production of F1 offspring. The precoital interval and gestational length were equivalent across all groups. There were also no changes across groups for the number of total implantation sites per litter and the number of total and live pups per litter at birth. There were also no significant differences across groups for percent postimplantation loss per litter or the number of dead pups at birth.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Treatment-related clinical observations of F0 males included rooting post-dosing in 5 males at 200 mg/kg/day. No other findings exhibited a treatment- or dose-related pattern of incidence or severity.
All 10 F0 females/group survived to scheduled sacrifice


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
No treatment-related effects on F0 male body weights were observed. Significantly higher weights for sd 7, 14, 21, and 28 (but not sd 0) occurred at 10 mg/kg/day, with no effects 100 or 200 mg/kg/day. F0 male body weight change was significantly increased at 10 mg/kg/day for sd 0-7 and unaffected for all other intervals at this dose and for all intervals at 100 and 200 mg/kg/day. No treatment-related effects on feed consumption were observed. Feed consumption values, expressed as g/day, were significantly increased at 10 mg/kg/day for sd 0 to 7, 7 to 14, and 0 to 14, with no effects at 100 or 200 mg/kg/day. There were no significant effects of treatment on F0 male feed consumption.

There were no significant differences among groups for F0 female body weights or body weight changes during the prebreed period (sd 0-14) or for the few females in the control and low-dose groups (10 mg/kg/day) that were not identified as sperm positive during the mating (sd 0-28) or postmating (sd 14-42) periods. There were no significant effects on F0 female feed consumption, expressed as g/day or g/kg/day, in any group during the 2 weeks of the prebreed period.


ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 males (10/group) were necropsied after 28 days of dosing (2 weeks prebreed + 2 weeks mating). There were no treatment-related effects on organ weights. Sacrifice body weights and absolute weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles with coagulating glands were all unaffected. Absolute prostate weight was significantly reduced at 100 mg/kg/day but statistically equivalent at 10 and 200 mg/kg/day. Organ weights relative to terminal body weights were unaffected for the thymus, heart, liver, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, and seminal vesicles plus coagulating glands. Relative brain weight was significantly reduced at 10 mg/kg/day and unaffected at 100 and 200 mg/kg/day. Relative prostate weight was significantly reduced at 10 and 100 mg/kg/day but unaffected at 200 mg/kg/day. Organ weights relative to brain weights were unaffected for the thymus, heart, spleen, paired kidneys, paired adrenal glands, paired testes, paired epididymides, prostate, and seminal vesicles with coagulating glands. Relative liver weight was significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS)
At scheduled necropsy of F0 females, there were no differences among groups for terminal body weights and organ weights (absolute, relative to terminal body weights, or relative terminal brain weights) for the brain, heart, liver, spleen, paired kidneys, paired adrenal ,lands, uterus with cervix and vagina, and paired ovaries. Absolute and relative (to both body and brain weights) weights of the thymus were significantly increased at 100 mg/kg/day and unaffected at 10 or 200 mg/kg/day.
No treatment-related gross necropsy findings were observed. No treatment related microscopic findings in any females (of 5/group) at 200 mg/kg/day were observed.



OTHER FINDINGS (PARENTAL ANIMALS):
FOB: No parameters evaluated in the FOB exhibited any statistically significant or biologically relevant difference across male F0 groups. There was also no effect of treatment on motor activity, auditory startle, or grip strength which were performed in week 4 prior to scheduled termination.
For female F0 groups the week 1 pupil size score was significantly reduced in the 200 mg/kg/day dose group and in week 3 the tail pinch score was also reduced in this dose group. No other parameters were affected at any time period in any dose group.

Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no indications of toxicity; no adult F0 parental toxicity in either sex, and no reproductive toxicity.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
There were 9, 7, 10, and 8 live litters on pnd 0 with 0, 10, 100, and 200 mg/kg/day, respectively. Live birth and stillbirth indices were unaffected, as were the survival indices for pnd 0-4, 4-7, 7-14, and 14-21 and the lactational index for pnd 4 (postcull) through 21. The mean number of live pups per litter for pnd 0, 7, 14, and 21 was unaffected across all groups. Mean F1 female and male anogenital distances (absolute or adjusted for body weight) per litter on pnd 0 were equivalent across all dose groups. Mean F1 pup body weights per litter (sexes combined or separately) were unaffected by treatment for all time points across all dose grou). Sex ratio (% males) per litter was also unaffected across all groups for all gestational intervals. There were no male pups in any litter with retained nipples on pnd 11-13. The number of areolae per pup and the number of pups with 1 or more areolae on pnd 11-13 were equivalent across all groups. F1 pup clinical observations during lactation indicated that the number of F1 pups found dead for pnd 0-21 was 2, 2, 5, and 2 pups at 0, 10, 100, and 200 mg/kg/day, respectively. In addition, 1 female pup at 100 mg/kg/day exhibited a herniated umbilicus on pnd 0.

Necropsy findings of F1 culled pups on pnd 4, and F1 pups found dead or euthanized moribund on pnd 0-21 were performed. Pups that died on pnd 0 exhibited closed ductus arteriosus (postnatal state), no air (fetal state) or air (postnatal state) in lungs, no or little milk in stomach, and autolysis of abdominal organs. Pups that died on pnd 1-21 included 1 at 100 mg/kg/day on pnd 1 with no findings and 1 pup on pnd 21 at 0 mg/kg/day that was cannibalized. These findings are typical of pups during early lactation and exhibited no treatment-or dose-related pattern. At weaning of the F1 litters on pnd 21, there were still 9, 7, 10, and 8 live litters at 0, 10, 100, and 200 mg/kg/day, respectively. There were 36, 23, 38, and 32 F1 male offspring and 32, 27, 41, and 27 F1 female offspring evaluated at scheduled sacrifice on pnd 21 for 0, 10, 100, and 200 mg/kg/day, respectively. There were no effects on F1 male or female body weights or organ weights at sacrifice on pnd 21 at any dose. Increases in relative thymus weight for females in the 100 and 200 mg/kg/day groups were not considered treatment related, because the absolute weights were not increased and thymus weight was not affected in F0 females or males. Necropsy findings for F1 male and female pups on pnd 21 included 1 male at 0 mg/kg/day with right undescended testis, with no findings in any other group and no findings in any female in any group.

Ten F1 males/group were necropsied as adults. There were no treatment-related effects observed for organ weights. Absolute paired kidney weights relative to body or brain weights) were significantly increased at 100 mg/kg/day and unaffected at 10 and 200 mg/kg/day. Liver weight, relative to terminal body weight), was significantly increased at 200 mg/kg/day.
There were no effects across all 4 groups for percent motile sperm, epididymal sperm concentration, testicular homogenization resistant SHC’s, sperm production (per testis), efficiency of daily sperm production (per gram of testis), or percent abnormal sperm. The % abnormal sperm values at 0 mg/kg/day and 200 mg/kg/daywere within historical control values.

The absolute weights and weights relative to terminal body and brain weights of the brain, thymus, heart, liver, spleen, paired kidneys, paired adrenals, paired ovaries, and uterus with cervix and vagina were equivalent across F1 female control and dose groups.

No histopathologic findings were identified in F1 males or females.

Prior to the scheduled sacrifice of the F0 females at the weaning of the F1 litters on pnd 21, auditory startle, motor activity and grip strength were assessed/ No differences among any parameters were identified.

CLINICAL SIGNS (OFFSPRING): Treatment related clinical observations in F1 males included rooting postdosing occurred in 2,2,7 and 9 F1 males at 0,10, 100 and 200 mg/kg/day, respectively. Salivating pre-/post dosing was observed in 4 males only at 100 mg/kg/day.
In F1 females, 7 females each in the 100 and 200 mg/kg/day exhibited rooting and 1,4 and 2 females in the 10, 100 and 200 mg/kg/day exhibited salivation post dosing, respectively.

BODY WEIGHT (OFFSPRING): Body weight change values were unaffected for all F1 male groups for all intervals from pnd 22 through 78. There were no differences in feed consumption for any postwean interal in any group.
On pnd 22, the mean F1 female body weight at 200 mg/kg/day was significantly reduced with no effects at any later time points at this dose or any time points at any other doses.

SEXUAL MATURATION (OFFSPRING): F1 male age at PPS was unaffected across all dose groups. F1 female age acquisition of vaginal patency was equivalent across all groups. Estrous cycle length monitored for the last 3 weeks of the post wean period for the F1 females, were equivalent across all the dose groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no F0 offspring toxicity during lactation in either sex and the acquisition of puberty in F1 males and females was unaffected. F1 postweanlings exhibited no systemic toxicity in either sex at any dose.
Reproductive effects observed:
not specified
Conclusions:
In conclusion, the test substance, administered by gavage once daily at 0, 10, 100, and 200 mg/kg/day to parental rats, from prebreed through mating, gestation, and lactation and direct dosing to F1 offspring from weaning to scheduled sacrifice, resulted in no adult F0 parental toxicity and no evidence of toxicity (systemic, reproductive, or developmental) in F1 offspring animals.
Therefore, the F0 male and female systemic NOAEL was at least 200 mg/kg/day. The F1 male and female systemic NOAEL was also at least 200 mg/kg/day. The NOAELs for F0 reproductive toxicity were at or above 200 mg/kg/day for males and females. The NOAELs for F1 offspring toxicity were also at or above 200 mg/kg/day for males and females.
Justification for read-across has been provided.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was conducted in accordance with a standardised guideline under GLP conditions. The quality of the database is therefore considered to be high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the key study, the repeated dose toxicity of 2,4,6 trimethyl phenol, a structural analogue of the registered material was investigated in a study conducted in accordance with the standardised guideline OECD 422 under GLP conditions. A subset of the animals tested was evaluated to a protocol similar or equivalent to OECD 407 (28 day repeat dose oral - rodent). 2,6-Xylenol and the structural analogue were determined to have sufficiently similar properties, such that available data on the structural analogue is considered to be suitable to address this endpoint. Due to the use of read across, the study was awarded a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Male and female Sprague-Dawley rats were exposed to the test material at concentrations of 0, 10, 100, 200 mg/kg/day in corn oil via oral gavage. F0 females were dosed premating through to the day prior to necropsy. Recovery males, females and 28 days females were dosed for 28 days. F1 animals were dosed post-weaning until the day before scheduled necropsy (duration of at least 7 weeks).

In the F0 generation, 10 males and 10 females were dosed, plus 5 additional animals per sex in the control and high dose groups which were designated recovery animals. In addition, 10 females (5 control and 5 in the high dose group) were designated as the 28 day females and were dosed for 28 days.

Parental animals were subjected to cage-side observations and detailed clinical observations. Body weight and food consumption were evaluated, as were reproductive indices.

There were no indications of toxicity (systemic, reproductive, developmental, or neurological) in the parental or F1 generation, the recovery males or females or the 28 day female dose groups evaluated in this study. The only effects were a post-dose rooting seen at the highest dose group. 

There were no significant effects of exposure to the test material on F0 mating, fertility, gestational, or pregnancy indices in F0 parental males and females during the production of F1 offspring. The precoital interval and gestational length were equivalent across all groups. There were also no changes across groups for the number of total implantation sites per litter and the number of total and live pups per litter at birth. There were also no significant differences across groups for percent post-implantation loss per litter or the number of dead pups at birth.

Under the conditions of this study the NOAEL for both the F0 and F1 generation was therefore determined to be at least 200 mg/kg bw.

An extended one-generation reproductive toxicity study is not required as there is sufficient weight of evidence from the OECD 422 screening assessment study on a structural analogue of 2,6-xylenol. In this study, rats were exposed to 10, 100 or 200 mg/kg/day of the structural analogue. No systemic, reprotoxic or developmental effects were observed over the course of the study in either the F0 parental animals (males or females) or the F1 progeny.

Moreover, as the substance is only used in closed systems no relevant long-term exposure will be observed. Based on these results it can be concluded that this endpoint has been adequately tested through the use of the screening study and that no additional studies are required.


Short description of key information:
A 28 day repeated dose/reprotoxicity study was carried out in rats in accordance with OECD 422 on a structural analogue of the registered material. The NOAEL was deemed to be greater than or equal to 200 mg/kg/day.

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information
A developmental toxicity study was carried out in rats in accordance with OECD 414. The NOEL for maternal toxicity was determined to be 60 mg/kg/day and the NOEL for developmental toxicity was determined to be 180 mg/kg/day. 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 29, 1996 to May 15, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study that was performed according to OECD Guideline 414.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CD-Crl: [SD] BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 64 days (at initiation of mating)
- Weight at study initiation: 195.1-255.4 g (Gestation Day 0)
- Housing: Individually, except during mating, in stainless steel suspended cages with wire mesh floors and fronts.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 46-72
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1996-07-23 To: 1996-08-23
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance were mixed with the corn oil to provide concentration levels of 12, 36, and 108 mg/mL for the low, mid and high dose groups, respectively. In addition, an appropriate amount of vehicle was dispensed for the control animals. Fresh dosing solutions were prepared at each concentration level weekly and dispensed for dosing daily. All dosing solutions were stored at ambient temperature. 5 mL/kg of body weight/day was administered to the animals. Initial dose volumes administered to individual animals were derived from Day 6 gestation body weights. The dose volumes were adjusted on days 9 and 12 of gestation to changes in individual animal body weights. Animals were dosed at approximately the same time each day.

VEHICLE
- Concentration in vehicle: 12, 36 and 108 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 125H0892, 56H0124
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to initiation of the study, mock batches of dosing solutions at the low and high levels were prepared. To assess homogeneity, three samples were collected from each of the top, middle and bottom portions of the containers with these formulations after mixing and analyzed. Formulations were considered homogeneous if the three samples at each level analyzed were within +/-10 % of each other and of the nominal concentration level. Stability of the test substance in the vehicle under ambient storage was established over a 14-day period at concentrations that ranged from 14 to 200 mg/mL. These analyses were performed in a pilot developmental toxicity study conducted by the laboratory. Since the concentration level for this study (i.e. 12 mg/ml) was outside this range, additional stability analyses were performed at this concentration only. Duplicate samples of the mock batch of solution prepared to assess homogeneity were assayed at 7 and 14 days post-preparation (Day 0 homogeneity assays, were used to establish the concentration at time of preparation).Dosing formulations for all three dose levels from the first three mixes used in study were assayed prior to use in the study (one sample per concentration was taken and analyzed in duplicate). The formulations were considered acceptable for use in study if the two samples from each concentration level were within 10% of each other.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1; The evenings for cohabitation of males with females were scheduled to provide female at Day 20 gestation sacrifice during the Monday-to-Friday work week.
- Length of cohabitation: Mating was conducted on 10 nights as follows: 23-27, 30 and 31 July and 1-3 August 1996.
- Proof of pregnancy: Vaginal smears were taken early in the morning following intervals of nightly cohabitation. Females were considered to have mated if sperm was noted microscopically in the vaginal rinse and/or a plug was observed in the vaginal opening. The day on which this evidence of mating was observed was defined as day 0 of gestation.
Duration of treatment / exposure:
Animals were treated once daily by oral gavage.
Frequency of treatment:
Animals were treated daily during the day 6-15 gestation interval.
Duration of test:
Animals were sacrificed on Day 20 of gestation.
Remarks:
Doses / Concentrations:
60, 180, and 540 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment (if not random): Females which mated were assigned to the groups daily in such a way as to provide an equal distribution of females among groups and equalize, as best possible, the Day 0 gestation mean body weights between groups.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: animals were observed for signs of pharmacologic or toxicologic effect and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0, 6-16, 18 and 20 of gestation; On days 6-15 of gestation, a detailed physical examination was conducted approximately one half hour after animals had been dosed.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 9, 12, 16, 18, and 20 of gestation; Day 20 gestation body weights were presented as actual and corrected (actual body weight minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined: Yes; Days 0-6, 6-9, 9-12, 12-16 and 16-20 (results reported as g/kg/day)


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Complete macroscopic postmortem examinations were performed on all test animals. External surfaces, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and paranasal sinuses, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs were examined. Gross lesions noted during the postmortem evaluations were retained and preserved in 10% neutral buffered formalin. The carcass of each female was discarded at completion of the postmortem evaluation. The livers were weighed for all females and liver/body weight ratios were calculated using the corrected Day 20 question weights.

Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes; The intact uterus (ovaries attached) was removed from the abdominal cavity. The ovaries were dissected from the uterus and evaluated. When no uterine implants were grossly apparent, the uterus was stained with ammonium sulfide to indentify foci of implantation and early foetal death. When no foci were visualized post staining, the female was considered not pregnant.
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes (total of fetuses plus resorptions)
- Number of early resorptions: Yes (evidence of implantation but no recognizable fetus)
- Number of late resorptions: Yes (recognizable dead fetus undergoing degeneration, regardless of size)
- Other: The number of live (movement to touch) and dead fetuses (lack of movement in response to touch with no visible degeneration) were measured.
Fetal examinations:
- External examinations: Yes: all per litter; Each fetus was individually identified, weighed, sexed externally (ano-genital distance) and given a gross examination for external malformation/variations to include observation for palatel defects.
- Soft tissue examinations: Yes: half per litter; The selected fetuses in each litter (alternating fetuses within the litter) were evaluated for malformation/variations using a microdissection procedure. Evaluations were performed on the fresh fetal specimens shortly after removal from the uterus. Fetuses designated for visceral evaluation were decapitated (head placed in appropriately labelled tissue bags and fixed in Bouin's solution for later evaluation). The fetal specimens were then secured beneath a dissecting microscope and dissected so as to permit evaluation of tissues in the thoracic and abdominal cavities.
- Skeletal examinations: Yes: half per litter; The remaining fetuses not used for soft tissue examinations in each litter were killed and the intact fetuses were eviscerated (internally sexed by inspection of the gonads) and processed for staining of the ossified skeletal structures. Fetal skeletal specimens were evaluated under a dissecting microscope (10-20X) for ossification variations and malformations.
- Head examinations: Yes: half per litter; Following a period of fixation, the fetal heads (see "soft tissue examinations" section above) were sectioned using a razor blade. The serial, traverse sections generated using this procedure were evaluated for malformation of the palate, eyes and brain under a dissecting microscope (10-20X). Following evaluation, head sections were placed in plastic cassettes for storage (one litter/jar) in a 10 % formalin solution.
Statistics:
See any other information on materials and methods including tables.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At the 540 mg/kg-bw/day dose group, 2/24 animals died (8.3% mortality), with both animals dying on day 10 of gestation. Excessive salivation was seen with increased frequency among the animals in the mid dose group during the treatment period. At the 540 mg/kg-bw/day dose group, the following findings were seen with increased frequency: lethargy, prostration, irregular gait, excessive lacrimation, labored breathing and staining of the skin/fur in the ano-genital area. Generally, these findings were most prevalent during the Day 6-10 gestation period. At the 180 mg/kg-bw/day dose group, a statistically significant reduction in food consumption was seen early in the treatment period (Days 6-9). For the remainder of the study, however, food consumption data for this group were comparable to control data. At the 540 mg/kg-bw/day dose group, food consumption was significantly lower than control throughout the treatment period. During the post treatment period food consumption of the 540 mg/kg-bw/day dose was statistically significantly increased in comparison to control data.
The mean liver to body weight ratio of the mid dose group was statistically significantly lower than controls with similar responses in liver to body weight ratio in the 540 mg/kg-bw/day dose group, this was not considered toxicologically significant.
No adverse effect of treatment was evident for maternal macroscopic postmortem examinations for any dose group. At the 540 mg/kg-bw/day dose group, 2/24 animals died for a mortality rate of 8.3%. Both animals died on day 10 of gestation. Excessive salivation was seen with increased frequency among the animals in the mid dose group during the treatment period. At the 540 mg/kg-bw/day dose high dose level, the following findings were seen with increased frequency: lethargy, prostration, irregular gait, excessive lacrimation, labored breathing and staining of the skin/fur in the ano-genital area. Generally, these findings were most prevalent during the Day 6-10 gestation period. Excessive salivation was also to the control group. Maternal body weight gains for the mid and 540 mg/kg-bw/day dose during the post-treatment period were comparable to control data with the exception of the 540 mg/kg-bw/day dose group which gained significantly more weight than control over days 16-18. Mean weight gains for the mid and high dose groups over the entire Day 6-20 gestation period using the corrected Day 20 gestation weights were lower than control and these differences were statistically significant.
As expected from the lower terminal body weights seen in the 180 and 540 mg/kg-bw/day dose groups, mean absolute liver weights for these groups were also significantly lower than controls. The mean liver to body weight ratio of the mid dose group was statistically significantly lower than controls with similar responses in liver to body weight ratio in the 540 mg/kg-bw/day dose group, this was not considered toxicologically significant. No adverse effect of treatment was evident for maternal macroscopic postmortem examinations for any dose group.
Dose descriptor:
NOEL
Effect level:
ca. 60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
ca. 180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOEL
Effect level:
ca. 540 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOEL
Effect level:
ca. 180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At the high dose level, mean fetal weights, distinguished by sex and as a composite for both sexes, were lower than control data. These differences were statistically significant and considered indicative of an adverse response to treatment. No adverse effect of treatment on fetal sex distribution data was observed for any dose level. No adverse effects of treatment were seen from fetal external examination data at any dose level. No malformations were seen during the fetal visceral examinations. A low incidence of commonly seen visceral variations was seen in the low dose group. No visceral variations were seen among the control, mid or high dose fetuses. The incidences of these findings on both a per fetus and per litter basis were well within the range of recent historical control data for the laboratory and in the absence of similar findings in fetuses from the higher dose levels, no adverse effect of treatment was indicated. No adverse effect of treatment at a dose level up to and including the mid dose was seen for the fetal skeletal malformation data. No skeletal malformations were seen in the mid or high dose fetuses. Likewise, no skeletal malformations were seen in the control fetuses. The only skeletal malformations seen were in the low dose group. The incidence of skeletal malformations in the low dose group was 2.6 % and the litter incidence was 16.7 %. These incidences did not differ statistically from control data. The incidences of fetuses with one or more ossification variations in the treated groups were comparable to or slightly lower than control data. The incidence of fetuses with at least one ossification for the low dose group differed statistically from control data but this was not considered indicative of an adverse response to treatment. The fetal and litter incidences for the different ossification variations seen during the study for the low and mid dose groups were considered comparable to control and not adversely affected by treatment. Only at the high dose was there a suggestion of retarded fetal ossification. This was indicated from the slight increases in fetal and litter incidences of reduced ossification of the cervical vertebral transverse processes and metatarsals for this group in comparison to control data.
Abnormalities:
not specified
Developmental effects observed:
not specified

Analytical Results:

Analyses of preliminary mixes at the low and high concentrations confirmed that the procedure used to prepare dosing solutions in this study produced homogenous mixtures and confirmed that the test substance at a concentration level of 12 mg/mL was stable in corn oil, under ambient storage conditions, for at least 14 days. Analyses conducted during the treatment period confirmed that dose solutions of appropriate concentrations were administered to the test animals. The mean analytical concentrations seen from the periodic analyses of dosing solutions used on study were as follows: 99.2 % (12.0 mg/ml), 100.6 % (36.0 mg/mL) and 102.3 % (108.0 mg/mL) of nominal.

Reprotoxicity Effects:

Pregnancy rates were comparable to controls. Actual pregnancy rates were: 91.7%, 100%, 95.8% and 95.8% for the control, 60, 180 and 540 mg/kg-bw/day dose groups, respectively. No adverse effects of treatment were evident from the number of corpora lutea, live fetuses, resorptions and uterine implantations per pregnant female. Likewise, the preimplantation loss indices and the ratio of resorptions to implants were comparable for treatment groups to the control.

Conclusions:
Mated female rats were administered 2,6-xylenol (60, 180 and 540 mg/kg/day) via oral gavage over the Day 6-15 gestation interval. Females were sacrificed on Gestation Day 20 and the maternal toxicity and developmental toxicity of the fetuses was evaluated. Based on the results of the study, the NOEL for maternal toxicity was determined to be 60 mg/kg/day and the NOEL for developmental toxicity was 180 mg/kg/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
180 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study was conducted in accordance with a standardised guideline under GLP conditions. The quality of the database is therefore considered to be high.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The potential of the test material to cause developmental toxicity effects was evaluated in a prenatal developmental toxicity study carried out in accordance with the standardised guideline OECD 414 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

CD-Crl: [SD] BR rats were dosed with the test material in corn oil via gavage at dose levels of 60, 180, and 540 mg/kg/day (24 per dose). Animals were treated daily during the day 6 to day 15 gestation interval.

Parental animals were subjected to cage-side observations and detailed clinical observations. Body weight and food consumption were evaluated. Animals were sacrificed on day 20 of gestation and a complete macroscopic postmortem examination performed on all test animals; ovary and uterine contents were examined.

Foetuses were subjected to external examinations, soft tissue examinations, skeletal examinations and head examinations.

Maternal toxicity was seen at the two highest dose levels, with some mortality and mean weight gains for the mid and high dose groups over the entire Day 6-20 gestation period being lower than control in a statistically significant fashion. This resulted in parental NOEL and LOEL values of 60 and 180 mg/kg/day, respectively.

Reduced foetal body weights were identified at maternally toxic doses, resulting in developmental NOEL and LOEL values of 180 and 540 mg/kg/day, respectively. No other effects were seen on the F1 generation with the exception that, at the high dose only, there was there a suggestion of retarded foetal ossification. This was indicated from the slight increases in foetal and litter incidences of reduced ossification of the cervical vertebral transverse processes and metatarsals for this group in comparison to control data.

Under the conditions of this study the NOEL for developmental toxicity was determined to be 180 mg/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one study available.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to reproductive and developmental toxicity.

Additional information

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