Phenol, C14-18-alkyl derivs.

Toxicity to reproduction

Administrative data

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:

12 December 2014 to 09 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C14-16-18 Alkyl phenol

EC Number:

931-468-2

Cas Number:

1190625-94-5

Molecular formula:

For substance information - see Section 1.2

IUPAC Name:

C14-16-18 Alkyl phenol

Test material form:

other: slightly viscous liquid

Details on test material:

- Appearance: Clear amber liquid
- Storage conditions of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:WI(Han) (outbred, SPF-Quality)
- Age at study initiation: (P) Approximately 11 weeks (males) or approximately 12 weeks (females)
- Weight at study initiation: (P) Males: 309 g; Females: 214 g
- Fasting period before study: No
- Housing: Pre-mating, animals were housed in groups of 5 animals/sex/cage in plastic cages (height 18 cm). During mating, females were caged together with males on a one-to-one-basis (plastic cages, height 18 cm). Post-mating, males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in plastic cages (height 18 cm). During the lactation period, pups were kept with the dam until termination. Sterilised sawdust was used as bedding material and paper as cage-enrichment/nesting material was supplied.
- Diet: ad libitum pelleted rodent diet
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: No data
To: 13 January 2015 (males); 26, 27 and 29 January 2015 and 06 and 09 February 2015 (females and/or pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.

DOSE VOLUME: 5 mL/kg bw

VEHICLE
- Specific gravity: 1.036

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: No data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; referred to as day 0 post coitum
- After successful mating each pregnant female was caged (how): Once mating occurred, the males and females were separated. Females were individually housed in plastic cages. During the lactation period, pups were kept with the dam until termination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF DOSE PREPARATIONS
Samples were stored and dispatched on dry ice to the test site for formulation analysis.
Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85 to 115 % of target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

SAMPLES
In total, 16 samples were included in this study, distributed over 4 formulation groups. The samples of the control group and the 75 mg/kg dose group were taken in duplicate from the middle position of the container and immediately stored on dry ice. Samples of treatment groups 25 and 225 mg/kg were taken in duplicate from the top, middle and bottom position of the container.
The samples were stored at a target temperature =-70 °C.

ANALYTICAL METHOD
- Analytical conditions
The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.
- Sample injections
The analytical run for the determination of the accuracy of preparation and homogeneity of the test material in formulations was acquired in the following order: analytical blank sample, calibration curve, analytical blank sample, analytical blank sample, procedural recovery sample high and low (replicate 1), analytical samples and procedural recovery samples high and low (replicate 2).
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were analysed by single injection.

FORMULAS
Response (Y): Peak area test material [mAU]

Calibration curve: Y = a + bX + cX²
where:
X = nominal concentration [mg/L]
c = curvature
b = slope
a = intercept

Analysed concentration (X): X = [-b + v(b² - 4c(a – y)) / 2c] x ((V x d) / w) [mg/g]
where:
w = weight sample [mg]
V = volume volumetric flask [mL]
d = dilution factor
[-b + v(b² - 4c(a – y)) / 2c] values are obtained from Analyst® version 1.5.2 (Applied Biosystems, Burlington, Canada).

Recovery: (Analysed concentration [mg/g] / Nominal concentration [mg/g]) x 100 [%]

Accuracy: (Analysed concentration [mg/g] / Target concentration [mg/g]) x 100 [%]

Relative difference (relative diff.): ((Ct - C0) / C0) x 100 [%]
where:
Ct = mean concentration of stored samples [mg/g]
C0 = mean concentration of non-stored samples [mg/g]

SPECIFICATIONS
- Run Acceptance
Calibration curves with a coefficient of correlation (r) of >0.99 and accuracies of the back calculated values of the calibration standard solutions in the range 85 to 115 % were accepted. Outliers could be discarded as long as minimally 75 % of the responses of the calibration were accepted.
The mean recovery of the procedural recovery samples at each level had to be in the range 85 to 115 %.
The response in all analytical blank samples at retention time of the test material should be less than or equal to 30 % compared to the response of the lowest calibration standard (mean response of the duplicate injections).
- Acceptance criteria formulations
Preparation of formulations was considered acceptable if the mean accuracy was in the range 85 to 115 % of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10 %.

RESULTS
- Calibration curves
Calibration curves were constructed using six concentrations. For each concentration, two responses were acquired. Regression analysis was performed using the least squares quadratic regression with a (1 / concentration²) weighting factor. The coefficient of correlation (r) was 0.9992.
The analytical blank samples injected before the calibration curve and after the highest calibrator showed responses at the retention time of the test material which were within the accepted range (lower than 30.0 % of the response of the lowest calibration standard).
- Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 85 to 115 %. It demonstrated that the analytical method was adequate for the determination of the test material in the test samples.
- Test samples
In the control group formulations, no test material was detected. The concentrations analysed in the formulations of the dose groups were in agreement with the target concentrations (i.e. mean accuracies between 85 and 115 %).
The formulations of the 25 and 225 mg/kg dose groups were homogeneous (i.e. coefficient of variation = 10 %).

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 75 and 225 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 28-day study with the test material in which dosages of 30, 100 and 300 mg/kg/day were administered by daily oral gavage.
No mortality occurred, and no toxicologically relevant clinical signs were noted. Body weights and food intake levels appeared unaffected by treatment. Haematological changes were confined to a slightly higher white blood cell count in females at 300 mg/kg/day. Clinical biochemistry changes included higher alanine aminotransferase and alkaline phosphatase activities, lower total protein and cholesterol levels, higher potassium and inorganic phosphate levels in males and/or females at 300 mg/kg/day. Necropsy revealed no apparent treatment-related lesions. Males and females at 300 mg/kg/day showed higher liver weights (absolute and/or relative; relative weight approximately 17 and 25 % higher than controls for males and females, respectively). Relative liver weights of females at 30 and 100 mg/kg/day also showed a statistically significant increase, but did not show a dose-related trend at these dosages.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily animals were examined for mortality/viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER: GENERAL REPRODUCTION DATA
- Parameters observed: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Sperm parameters (parental animals):

Parameters examined in [all] male parental animals: testis weight and epididymis weight and staging of spermatogenesis.

Litter observations:

PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
-Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
The animals were not deprived of food overnight. All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane and subsequently exsanguinated.
- Male animals: Males were necropsied following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: Females which delivered were necropsied on lactation Days 5 to 7. Females which failed to deliver (nos. 54 and 74) were necropsied on post-coitum Days 26 and 27 (females with evidence of mating).

GROSS NECROPSY
All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following tissues and organs were collected and fixed in 10 % buffered formalin (neutral phosphate buffered 4 % formaldehyde solution): cervix, clitoral gland, coagulation gland, epididymides, female mammary gland area, kidneys, liver, ovaries, preputial gland, prostate gland, seminal vesicles, testes, stomach, uterus, vagina and all gross lesions.
The epididymides and testes were fixed in modified Davidson's solution and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.
In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Testes and epididymides weight and terminal body weight was recorded for all parental males on the scheduled day of necropsy.

All organ and tissue samples were processed, embedded and cut at a thickness of 2 to 4 µm. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.

The following slides were examined by a pathologist:
- Ovaries, testes, epididymides, stomach, kidneys and liver of the animals of the control and 224 mg/kg dose groups.
- Stomach (both sexes) and kidneys (males) of all 25 and 75 mg/kg dose group animals based on (possible) treatment-related changes in these organs in the high dose group.
- The additional slides of the testes of the selected 5 males of the control and 224 mg/kg dose groups and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups, i.e. female/male numbers 54/14 (25 mg/kg dose group) and 74/34 (225 mg/kg dose group). Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on Days 5 to 7 of lactation. Pups found dead during the weekend were fixed in identified containers containing 70 % ethanol.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. Relevant abnormalities were collected and fixed in 10% buffered formalin. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. Also if possible, defects or cause of death were determined.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data. Test statistics were calculated on the basis of exact values for means and pooled variances.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = (Number of pregnant females / Number of paired females) x 100
Conception index (%) = (Number of pregnant females / Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

For each group, the following calculations were performed:
Percentage live males at first litter check = (Number of live male pups at first litter check / Number of live pups at first litter check) x 100
Percentage live females at first litter check = (Number of live female pups at first litter check / Number of live pups at first litter check) x 100
Percentage of postnatal loss = (Number of dead pups before planned necropsy / Number of live pups at first litter check) x 100
Viability index = (Number of live pups before planned necropsy / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period.

CLINICAL SIGNS (PARENTAL ANIMALS)
No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing for animals of the 25, 75 and 225 mg/kg/day dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste/irritant potential of the test material rather than a sign of systemic toxicity.
Hunched posture and pale faeces noted at 225 mg/kg/day on one or two days for one male and female each, respectively, was considered not toxicologically relevant given the low incidence observed and inconsistent occurrence with continuing treatment.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend.

BODY WEIGHTS (PARENTAL ANIMALS)
Males at 225 mg/kg/day showed a slightly lower mean body weight and body weight gain from Day 8 of the premating period onwards, achieving a level of statistical significance for body weight on Day 8 of premating and for body weight gain on all occasions. Total weight gain was approximately 5 % lower than in controls. Females treated at 225 mg/kg/day had slightly lower body weights during the post-coitum and lactation periods, though the only statistically significant difference from controls was seen on Day 1 of lactation. Body weight gain of these females was similar to control levels throughout treatment.
Body weight and body weight gain at 25 and 75 mg/kg/day was similar between treated and control animals.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Males treated at 225 mg/kg/day showed an apparent lower absolute and relative food consumption during the first week of the premating period. Females treated at 225 mg/kg/day also showed an apparent lower relative food consumption during the first week of the premating period. These females also showed slightly lower absolute and relative food consumption intermittently during the post-coitum phase but achieved a level of statistical significance on a single occasion only (Days 7 to 11).
One female at 225 mg/kg/day (no. 71) had a low absolute and relative food intake during lactation, which was considered to be related to the high post-natal loss of this animal. Also, since other females of this dose group showed food intake levels that were comparable to control levels, this was considered not toxicologically relevant.
The statistically significant higher absolute and relative food consumption during lactation for females treated at 75 mg/kg/day occurred in the absence of a dose-related trend and was therefore considered to be unrelated to treatment with the test material.
Food consumption before or after allowance for body weight at 25 mg/kg/day was similar between treated and control animals.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test material-related macroscopic findings.
The recorded macroscopic findings were considered to be spontaneous in nature and did not distinguish treated animals from the controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test material-related microscopic findings after treatment were noted in the forestomach and kidneys (males only).
These test material-related findings in the forestomach were observed in 3/10 females of the 75 mg/kg/day group and in 8/10 males and 6/10 females of the 225 mg/kg/day group and consisted of:
- Erosion/ulceration in 1/5 females (1 minimal) of the 225 mg/kg/day group.
- Squamous cell hyperplasia in 2/10 females (2 minimal) of the 75 mg/kg/day group and in 8/10 males (6 minimal, 2 slight) and 6/10 females (3 minimal, 3 slight) of the 225 mg/kg/day group.
- Lymphogranulocytic sub-epithelial inflammation in 3/10 females (2 minimal, 1 slight) of the 75 mg/kg/day group and in 8/10 males (7 minimal, 1 slight) and 5/10 females (4 minimal, 1 slight) of the 225 mg/kg/day group.
- Submucosal oedema in in 2/10 females (2 minimal) of the 75 mg/kg/day group and in 1/10 males (1 minimal) and 1/10 females (1 slight) of the 225 mg/kg/day group.
The forestomach findings of female number 58 of the 25 mg/kg/day group were focal and showed a different type of response compared to the 75 and 225 mg/kg/day treated rats (granulomatous instead of lymphogranulocytic inflammation) and were in general more severe. The remainder of the forestomach was unaffected. Therefore, these findings in this single rat were considered to be incidental and unrelated to the treatment with the test material.
Hyaline droplet accumulation was observed at an increased severity and/or incidence in the kidneys of treated males. These findings were noted in 8/10 males of the 25 mg/kg/day group (5 minimal, 3 slight), in 5/10 males of the 75 mg/kg/day group (3 slight, 2 moderate) and in 9/10 males of the 225 mg/kg/day group (5 minimal, 2 slight, 2 moderate), compared to in 6/10 males of the control group (5 minimal, 1 slight).
There were no other test material-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test material-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were 1/10 couples treated at 25 mg/kg/day (numbers 14 & 54) and 1/10 couples treated at 225 mg/kg/day (numbers 34 & 74) without offspring. No abnormalities were seen in the reproductive organs which could account for their lack of offspring.
Furthermore, spermatogenic staging profiles were normal for all males examined.
The lack of offspring in those two couples without test material-related morphologic alterations was considered to be unrelated to treatment.

DISCUSSION
Treatment resulted in no systemic toxicity but local forestomach lesions noted at 75 mg/kg/day (females) and 225 mg/kg/day (both sexes), which consisted in general of multifocal to diffuse hyperplasia of the squamous epithelium (sometimes accompanied by hyperkeratosis), lymphogranulocytic subepithelial inflammation and/or (sub)mucosal oedema. In a single female of the 225 mg/kg/day group, erosion/ulceration was additionally noted. These findings may reflect a response to damage of the forestomach epithelium by the test material, including an interruption of the protective forestomach epithelium (erosion/ulceration and inflammatory responses around the junction epithelium-lamina propria). As such, these findings were considered adverse in nature.
At 225 mg/kg/day, the slightly lower body weight/body weight gain and absolute and/or relative food consumption of males and females was determined to be not adverse given that these changes were considered mild in nature and/or were not consistently seen with continuing treatment. It could not be excluded that these slight changes in body weight and food intake were related to the aforementioned forestomach lesions.
The hyaline droplet accumulation observed in this study at an increased severity and/or incidence in the kidneys of treated males is considered to represent alpha-2u-globulin. This is a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. In absence of a clear dose-response and other morphologic hallmarks of kidney damage, the hyaline droplet accumulation is not considered to be an adverse finding. Moreover, this male rat specific protein is not present in female rats or in higher mammals, including man.

REPRODUCTION/DEVELOPMENTAL DATA (PARENTAL ANIMALS)
- Reproduction data
Reproductive parameters (mating, fertility and conception indices, pre-coital time, and number of corpora lutea and implantation sites) were considered unaffected by treatment.
One female each at 25 and 225 mg/kg/day (numbers. 54 and 74, respectively) did not become pregnant.
The incidence of non-pregnancy showed no dose-related trend, and was considered to be normal for rats treated under the conditions of this study.
- Developmental data
Developmental parameters (gestation index and duration, parturition, maternal care and early postnatal pup development (sex ratio, mortality, clinical signs, body weight and macroscopy)) were considered unaffected by treatment.
- Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

- local

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the presence of adverse forestomach lesions starting at 75 mg/kg/day in females and 225 mg/kg/day in males

2

Dose descriptor:

NOAEL

Remarks:

- systemic

Effect level:

225 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

3

Dose descriptor:

NOAEL

Remarks:

- reproductive toxicity

Effect level:

225 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT (OFFSPRING)
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were considered unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The statistically significantly lower sex ratio at 225 mg/kg/day was considered to be unrelated to treatment since a similar, but opposite ratio was noted for control litters.

MORTALITY (OFFSPRING)
Six pups of the control group, six pups at 25 mg/kg/day, five pups at 75 mg/kg/day and 10 pups at 225 mg/kg/day (of which eight were from a single litter (no. 71)) were found dead or went missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups surviving until scheduled necropsy consisted of a blue spot in the neck or on the head, small tail, a wound on the right flank and lean appearance. One pup of litter 59 (25 mg/kg/day) that was sacrificed in extremis showed absence of milk in the stomach, a deformed head and bent tail. One pup of litter 71 (225 mg/kg/day) that was found missing on Day 6 had a lean appearance and reduced milk content in the stomach on the previous day. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and showed no apparent dose-related trend. These findings were therefore considered to be unrelated to treatment.

BODY WEIGHTS (OFFSPRING)
Body weights of pups were unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included autolysis and absence of milk in the stomach. One pup of litter 59 (25 mg/kg/day) that was sacrificed in extremis also showed a deformed head and bent tail. A small tail was noted for one surviving pup from another litter. The nature and incidence of these findings remained within the range considered normal for pups of this age, and showed no apparent dose-related trend. These findings were therefore considered to be unrelated to treatment.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 225 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of Reproduction data

	Dose Group (mg/kg)
	0	25	75	225
Females paired	10	10	10	10
Females mated	10	10	10	10
Females pregnant	10	9	10	9
Females with living pups on Day 1	10	9	10	9
Mating index (%)	100	100	100	100
Fertility index (%)	100	90	100	90
Conception index (%)	100	90	100	90
Gestation index (%)	100	100	100	100

 

Table 2: Number of Females Mated on Each Day During the Pairing Period

Day of the Pairing Period	Dose Group (mg/kg)
	0	25	75	225
1	1	2	1	4
2	2	-	5	3
3	1	2	1	2
4	6	5	2	1
12	-	1	-	-
13	-	-	1	-
Median pre-coital time (days)	4	4	2	2
Mean pre-coital time (days)	3.2	4.0	3.5	2.0

Table 3: Summary of Corpora Lutea and Implantation Sites

Parameter		Dose Group (mg/kg)
		0	25	75	225
Corpora lutea	Mean SD N	13.7 3.3 10	14.1 1.5 9	14.2 1.2 10	12.4 1.3 9
Implantations	Mean SD N	12.5 3.7 10	12.9 2.3 9	13.9 1.3 10	11.1 2.0 9

SD = standard deviation

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 25 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 225 mg/kg/day was derived.

Executive summary:

The potential of the test material to cause reproductive toxicity was investigated in a reproduction/developmental toxicity screening test conducted in accordance with the standardised guidelines OECD 421 and US EPA OPPTS 870.3550 under GLP conditions.

The test material, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats at dose levels of 0, 25, 75 and 225 mg/kg/day. Each group consisted of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42 to 56 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum and during at least 4 days of lactation.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, pre-coital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analysed once during the study to assess accuracy and homogeneity. The analysis showed that the formulations were prepared accurately and were homogenous.

- Parental results

Treatment resulted in no systemic toxicity but local forestomach lesions noted at 75 mg/kg/day (females) and 225 mg/kg/day (both sexes), which consisted in general of multifocal to diffuse hyperplasia of the squamous epithelium (sometimes accompanied by hyperkeratosis), lymphogranulocytic sub-epithelial inflammation and/or (sub)mucosal oedema. In a single female of the 225 mg/kg/day group, erosion/ulceration was additionally noted. These findings may reflect a response to damage of the forestomach epithelium by the test material, including an interruption of the protective forestomach epithelium (erosion/ulceration and inflammatory responses around the junction epithelium-lamina propria). As such, these findings were considered adverse in nature.

At 225 mg/kg/day, the slightly lower body weight/body weight gain and absolute and/or relative food consumption of males and females was determined to not be adverse given that these changes were considered mild in nature and/or were not consistently seen with continuing treatment. It could not be excluded that these slight changes in body weight and food intake were related to the aforementioned forestomach lesions.

The hyaline droplet accumulation observed in this study at an increased severity and/or incidence in the kidneys of treated males is considered to represent alpha-2u-globulin. This is a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. In absence of a clear dose-response and other morphologic hallmarks of kidney damage, the hyaline droplet accumulation is not considered to be an adverse finding. Moreover, this male rat specific protein is not present in female rats or in higher mammals, including man.

No clinical signs of toxicity were noted during the observation period. Testes and epididymides weights were unaffected by treatment.

- Reproductive results

No reproduction toxicity was observed up to the highest dose level tested (225 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, pre-coital time, numbers of corpora lutea and implantation sites and histopathological examination of reproductive organs).

- Developmental results

No developmental toxicity was observed up to the highest dose level tested (225 mg/kg/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

Under the conditions of this study, a parental local No Observed Adverse Effect Level (NOAEL) of 25 mg/kg/day was derived based on the presence of adverse stomach lesions. A parental systemic, reproduction and developmental NOAEL of at least 225 mg/kg/day was derived.