Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 10 weeks, females 13 weeks
- Weight at study initiation: males 235 to 307 g; females 199 to 246 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA) without cage enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: males 8 days, females 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed
that would interfere with the objectives of the study. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Fac
ility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-21.6
- Humidity (%): 45.4-70.0
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 March 2017 To: 01 June 2017

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) for a maximum of 8 days until use, if not used on the day of preparation.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or sperm in the vaginal lavage referred to as day 0 post-coitum.
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.
-concentration analysis
Duplicate samples (approximately 5 g) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
-homogeneity analysis
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. After acceptance of the analytical results, backup samples were discarded.
-stability analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the experimental conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated for 28 days, up to and including the day before scheduled necropsy.
Females that delivered offspring were treated for 50 - 55 days; Females that failed to deliver healthy offspring were treated for 41 days.

Frequency of treatment:

continuous (by feed)

Dose / conc.:

500 other: ppm (nominal 30 mg/kg bw)

Dose / conc.:

2 000 other: ppm (nominal 130 mg/kg bw)

Dose / conc.:

7 500 other: ppm (nominal 500 mg/kg bw day)

No. of animals per sex per dose:

10 (and 3 additional high dose males to determine sperm parameters after 4 days of administration)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the chemical properties of the test item which becomes an acid upon administration because the ester is cleaved with the potential for acidification of the blood, on the results of a 14-day oral range finding study with dietary administration of Veramoss in the rat, and in an attempt to produce graded responses to the test item.
In the 14 day study the NOAEL was 7500 ppm corresponding to 758 mg/kg bw/day for males and 735 mg/kg bw/day for females which was the highest dose level tested (in consultation with the Sponsor). No toxicologically significant changes were noted in clinical appearance, body weight, food consumption, macroscopic examination, organ weights and microscopic examination.

Parental animals: Observations and examinations:

- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations:on the first day of administration, and weekly thereafter. Mated fe
males were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4
, 7, and 13. A fasted weight was recorded on the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g
food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain
data: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted
averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal
was maintained during the study, but no quantitative investigation was introduced as no effect was
suspected.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters according to Guidelines were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters according to guidelines were examined.

URINALYSIS: No

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 15 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight

the following assessments were performed after four days of dietary administration for additional males in high dose (3 in total):
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy.
Sperm motility was assessed and sperm smears for morphological evaluation were fixed from all samples. Abnormal forms of sperm from a differential count of 200 spermatozoa (if possible) per animal was recorded.
For all Group 4 additional males one testis and one epididymis (left) were removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers.
For all Group 4 additional males one testis and one epididymis (right) were removed, placed in labeled bags for possible future histopathology evaluation. These will be discarded at finalization of the report or after approval from the Sponsor.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); Blood samples were collected from two of the surplus pups (if possible from one male and one female pup).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
mortality, clinical observations, body weights, sex, anogenital distance, areola/nipple retention (males), plasma T4 measurement PND 14-15 pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (28 days of administration); Group 4 additional males after 4 days of treatment.
- Maternal animals: All surviving animals on PND 15-16; Females which failed to deliver (with evidence of mating: post-coitum days 26-27)

GROSS NECROPSY
- Gross necropsy according to Guideline

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to Guideline.

Postmortem examinations (offspring):

GROSS NECROPSY
Pups were euthanized on PND 14-15. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for blood sampling were the same pups as selected for thyroid preservation.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): Total number of offspring born /Total number of uterine implantation sites) x 100
Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Offspring viability indices:

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
Viability index (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

Two females were sacrificed unscheduled on PND 1 (one Group 1 and one Group 4 female) due to aggressive behavior and cannibalism of their healthy pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Females at 500 ppm and 7500 ppm showed somewhat lower body weight gain during the post-coitum period (statistically significant on Day 14 and Day 17 for 500 ppm females and Day 14 for 7500 ppm females), resulting in 5% and 7% lower mean body weights on Day 20 post-coitum (not statistically significant) respectively. Thereafter, during lactation, mean body weights of 7500 ppm females remained lower compared to controls (up to 10%), reaching statistical significance at lactation Days 4
and 7 (the difference on lactation Day 13 was smaller and not statistically significant due to the poor weight gain of control females between Days 7-13). Body weight gain of 7500 ppm females during lactation was normal. Body weight and body weight gain of females treated up to 2000 ppm and males treated up to and including 7500 ppm were unaffected by treatment.
The statistically significant increase in body weight gain noted in females at 2000 ppm during lactation (Day 13) were considered unrelated to treatment due to the lack of a dose-related response and/or relatively low control value.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted. At 7500 ppm, food consumption of males was higher compared to controls on several days during the treatment period, resulting in a higher overall mean food consumption for the premating phase (relative difference from controls: 15%). Females at 7500 ppm consumed less food than controls on the first treatment day. Thereafter, their food consumption generally remained close to control values (for one cage of the 7500 ppm females (cage no. 18) food consumption values were higher compared to controls on a few days during the pre-mating period). Overall, food consumption was considered not to be affected by treatment since no consistent differences from controls were noted. The statistically significant differences in females at 2000 and 7500 ppm noted on a single occasion during the postcoitum period (Days 22-23 when group means were based on only a few females) and the lactation period (Days 9-10) and for 7500 ppm the food consumption corrected for body weight in the lactation period (Days 12-13) were considered to be chance findings.

The mean daily intake of the test item per kg body weight during the different phases of the study is given by `Any other information on results incl tables`.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters (red and white blood cells) were considered not to have been affected by treatment.
Isolated statistically significant variations noted in red blood cell parameters (slightly lower hemoglobin and hematocrit in females at 500 and 7500 ppm) were considered unrelated to treatment due to the absence of a dose-related trend.
Coagulation parameters were considered not to have been affected by treatment.
An isolated statistically significant variation noted in prothrombin time (lower in males at 2000 ppm) was considered unrelated to treatment due to the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant differences were noted between the 7500 ppm group and the control group:
• Higher potassium in males (1.09 x of control). Values in 7500 ppm males and in one control male were at or slightly above the upper limit of the historical control range. (Historical control data period 2015-June 2017; potassium in male rats (mmol/L) mean = 3.94, P5-P95 = 3.54-4.37 (n=219))
• Lower total protein in females (0.92 x of control). Values in 7500 ppm females remained within the historical control range. (Historical control data period 2015-June 2017; total protein in female rats (g/L) mean = 61.8, P5-P95 = 54.7-68.1 (n=186))
In females a large variety in bile acids were noted, both in the control group and 7500 ppm females this is caused by an individual animal with a markedly higher value compared to the remainder of the group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. In the absence of a dose-related response, the statistically significantly lower fore limb grip strength in females at 500 ppm was considered unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to be affected by treatment.
Most females had regular cycles, generally of four days. Two treated females had an irregular cycle during the pre-mating period (one low dose and one high dose female), and for one high dose female cycle length during treatment could not be determined (her vaginal smears showed met-estrus on the fifth treatment day and di-estrus on ten successive days thereafter). These females had normal litters. The cycle abnormalities were considered unrelated to treatment due to their incidental occurrence and lack of a dose-related trend.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

To evaluate the sperm analysis results, the results obtained for the three additional males treated for four days at 7500 ppm were compared with historical control data (the study did not include sperm analysis on concurrent controls). It should be remarked that the small number of males examined and lack of concurrent control data precludes definite conclusions on possible acute effects (or lack thereof) of the test item on sperm parameters.
Sperm motility (% motile sperm and % progressive motility) appeared normal in 2/3 males and was zero in one male. Zero motility is incidentally seen in untreated control males.
Testicular sperm counts were lower compared to the historical control group mean but remained in the normal range. The same was true for epididydimal sperm count in one male whereas epididydimal sperm counts in the other two males were comparable to the historical control mean.
Examination of sperm morphology showed relatively high numbers of sperm with detached head in all three males.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 2/10 couples of the 500 ppm group and 2/10 couples of the 2000 ppm group which had no offspring. No abnormalities were seen in the reproductive organs which could account for their lack of offspring.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. No treatment related effects were observed on mating index, precoital time, number of implantation sites and fertility index.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 717 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

overall mean test item intake

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

overall mean test item intake

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed incidentally in treated pups remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment. It was noted that four pups of a control female had no milk in their stomach on PND 1 (three were found dead at first litter check, one was lethargic and sacrificed in extremis). The three surviving pups of this female had less milk in their stomach at first litter check (two were dehydrated the next day and one was skinny on PND 3-4). These pup findings were likely to be related to the considerably reduced food consumption of the female at the end of gestation and the first day of lactation (on the other days, her food intake was normal).
Most pups of another control female had scabs on their snout (up to PND 5) and three of her pups showed signs of cannibalism (one found dead at first litter check, two sacrificed in extremis due to cannibalism by the dam). Another pup went missing, presumably cannibalized, on PND 3. That female showed no clinical signs of toxicity, but her food consumption was considerably reduced throughout gestation and lactation.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 87, 99, 99 and 90% in Groups 1, 2, 3 and 4, respectively.
Postnatal loss in Groups 1 and 4 was relatively high (total number of pups lost: 15 and 11, respectively). This was due to the high loss in two litters due to abnormal behavior of the dams (dams cannibalized healthy pups which was unrelated to treatment). These females were sacrificed on PND 1 together with their healthy pups (all 10 pups of the control female and six surviving pups of the high dose female). The other pups of the high dose female went missing (one pup, presumably cannibalized) or died spontaneously on PND 1 (three pups showed bite marks at macroscopic examination). A few other females of control and high dose lost one or a few pups which went missing (presumably cannibalized) on PND 2-3 (one pup of two control females and one high dose female) or were sacrificed in extremis on PND 1 (one pup and two pups of two control females). In the intermediate dose groups, only one low dose pup and one mid dosepup went missing (presumably cannibalized) on PND 2. This postnatal loss was considered to be unrelated to treatment as the incidence showed no doserelated trend.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be adversely affected by treatment.
Body weight gain of pups at 7500 ppm tended to be slightly lower than that of control pups, resulting in about 7% lower mean body weights on PND 13. As statistical significance was not achieved and the difference was only slight, this finding was considered not to be toxicologically relevant.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

pups were sacrificed PND 14-15

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-15 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Description (incidence and severity):

pups were sacrificed PND 14-15

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
Pups that survived until scheduled sacrifice showed no abnormalities at macroscopic examination. The nature and incidence of macroscopic findings noted in pups that were found dead or sacrificed on PND 1 remained within the range considered normal for pups of this age or occurred in pups of the control group, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Treatment up to and including 7500 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed on PND 13.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 500 other: ppm; converted to 1175 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Treatment related:

no

Diet analyses

Accuracy: No test item was detected in the control diet.

The concentrations analyzed in the 500, 2000 and 7500 ppm diets were in agreement with target concentrations, with mean accuracies between 97% and 100%).

Homogeneity: The 500 ppm and 7500 ppm diets were homogeneous (coefficient of variation 2.9 and 5.0%, respectively).

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the text table below.

		Mean over means intake[mg test item/kg body weight/day] (mean range indicated between brackets)
sex	Study period	500 ppm	2000 ppm	7500 ppm
Males	Pre-mating	45 (41-51)	197 (168-238)	790 (623-1062)
	Post-mating	35 (31-47)	150 (134-238)	590 (500-819)
	Mean of means^a	41	181	717
Females	Pre-mating	42 (36-59)	177 (145-238)	737 (471-1035)
	Post-coitum	64 (44-76)	235 (116-296)	995 (648-1217)
	Lactation	113 (73-144)	472 (305-593)	1907 (1116-2514)
	Mean of means^a	71	281	1175

aMean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14x mean pre-mating) + (13x mean post-mating)) / 27

Females (Group 2 and 3): ((14 x mean pre-mating) + (26 x mean post-coitum) + (14 x mean lactation)) / 54

Females (Group 4): ((14 x mean pre-mating) + (23 x mean post-coitum) + (14 x mean lactation)) / 51

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of at least 7500 ppm, which corresponds to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively. In view of the high intake values being close or exceeding 1000 mg/kg bw the conclusion for repeated dose, fertility and developmental toxicity is that no adverse effects are seen.

Executive summary:

In this executive summary the repeated dose toxicity is presented is the same as in the repeated dose section. In addition, to fertility also the developmental toxicity is presented in this section.

Introduction: The objectives of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) were to determine the potential toxic effects of the substance when given via diet for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) no-observed-adverse-effect levels (NOAELs) were evaluated. In this section only the repeated dose parameters are presented. The fertility and developmental effects are presented in the reproductive toxicity sections.

Method: The dose levels in this study were based on the results of a dose range finder. Rats (10/sex) received food with vehicle or test substance at levels of 500, 2000 or 7500 ppm (nominal 30, 130 and 500 mg/kg bw/day, respectively).

Results: Accuracy and homogeneity of diet preparations were demonstrated by analyses. The intake of the substance was higher than expected and resulted in an overall average for the high dose of 717 mg/kg bw for males and 1175 mg/kg bw for females.

Clinical signs: Exposure to the test item up to 7500 ppm was well tolerated as indicated by the absence of adverse changes in the parental parameters examined in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations).

Body weight gainof females at 7500 ppm was somewhat reduced during lactation. As the associated decreases in mean body weight did not exceed 10% this finding was considered not to be adverse.

Heamatology: No adverse effect seen.

Clinical biochemistry: No adverse effects seen. A higher mean plasma level of potassium was noted in males treated at 7500 ppm. This change was regarded as non-adverse based on its modest magnitude (9% difference from controls) and the absence of changes indicative of target organ toxicity or alterations in the general function of the animals. A lower mean plasma level of total protein was observed in females treated at 7500 ppm. This change was regarded as non-adverse due to its slight magnitude (8%, within the historical control range, see results section) and the absence of any evidence of conditions known to be associated with decreases in total protein.

Organ effects: Absolute and relative weights, macroscopically and microscopically there were no adverse effect seen.

Fertility: No reproduction toxicity was observed up to the highest dose level tested (7500 ppm).

No treatment-related changes were noted in any of the routine reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrus cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental toxicity: No developmental toxicity was observed up to the highest dose level tested (7500 ppm). No treatment-related or toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-15 pups, and macroscopic examination.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of at least 7500 ppm was established, which corresponds to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental no-observed-adverse-effect level

(NOAEL) of at least 7500 ppm was established, which corresponds to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively. In view of the high intake values being close or exceeding 1000 mg/kg bw the conclusion for repeated dose, fertility and developmental toxicity is that no adverse effects are seen.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

2008

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

EC Number:

225-193-0

EC Name:

Methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Cas Number:

4707-47-5

Molecular formula:

C10H12O4

IUPAC Name:

methyl 2,4-dihydroxy-3,6-dimethylbenzoate

Test material form:

other: solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males 10 weeks, females 13 weeks
- Weight at study initiation: males 235 to 307 g; females 199 to 246 g
- Fasting period before study: no
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA) without cage enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Diet (e.g. ad libitum): free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: males 8 days, females 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-21.6
- Humidity (%): 45.4-70.0
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 March 2017 To: 01 June 2017

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C) for a maximum of 8 days until use, if not used on the day of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by using a validated analytical procedure.
-concentration analysis
Duplicate samples (approximately 5 g) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
-homogeneity analysis
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. After acceptance of the analytical results, backup samples were discarded.
-stability analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the experimental conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Males were treated for 28 days, up to and including the day before scheduled necropsy
Females that delivered offspring were treated for 50 - 55 days; Females that failed to deliver healthy offspring were treated for 41 days.

Frequency of treatment:

continuous (by feed)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 other: ppm (equivalent to 30 mg/kg bw nominal)

Doses / concentrations 2

Dose / conc.:

2 000 other: ppm (equivalent to 130 mg/kg bw nominal)

Doses / concentrations 3

Dose / conc.:

7 500 other: ppm (equivalent to 500 mg/kg bw, nominal)

No. of animals per sex per dose:

10 (and 3 additional high dose males to determine sperm parameters after 4 days of administration)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the chemical properties of the test item which becomes an acid upon administration because the ester is cleaved with the potential for acidification of the blood, on the results of a 14-day oral range finding study with dietary administration of Veramoss in the rat, and in an attempt to produce graded responses to the test item.
In the 14 day study the NOAEL was 7500 ppm corresponding to 758 mg/kg bw/day for males and 735 mg/kg bw/day for females which was the highest dose level tested (in consultation with the Sponsor). No toxicologically significant changes were noted in clinical appearance, body weight, food consumption, macroscopic examination, organ weights and microscopic examination.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:on the first day of administration, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters according to Guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters according to guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: week 4 of treatment (males) or last week of lactation (females)
- How many animals: 5 per group
- Dose groups that were examined: all
- Parameters checked: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength and locomotor activity.

OTHER: sperm analysis from group 4 additional males after 4 days of dietary administration; plasma levels of T4 in F0 males and PND 14-15 pups, estrous cycle determination F0 females.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to Guideline

HISTOPATHOLOGY: Yes, according to Guideline

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
non-parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

Two females were sacrificed unscheduled on PND 1 (one Group 1 and one Group 4 female) due to aggressive behavior and cannibalism of their healthy pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Females at 500 ppm and 7500 ppm showed somewhat lower body weight gain during the post-coitum period (statistically significant on Day 14 and Day 17 for 500 ppm females and Day 14 for 7500 ppm females), resulting in 5% and 7% lower mean body weights on Day 20 post-coitum (not statistically significant) respectively. Thereafter, during lactation, mean body weights of 7500 ppm females remained lower compared to controls (up to 10%), reaching statistical significance at lactation Days 4 and 7 (the difference on lactation Day 13 was smaller and not statistically significant due to the poor weight gain of control females between Days 7-13). Body weight gain of 7500 ppm females during lactation was normal. Body weight and body weight gain of females treated up to 2000 ppm and males treated up to and including 7500 ppm were unaffected by treatment.
The statistically significant increase in body weight gain noted in females at 2000 ppm during lactation (Day 13) were considered unrelated to treatment due to the lack of a dose-related response and/or relatively low control value.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted.
At 7500 ppm, food consumption of males was higher compared to controls on several days during the treatment period, resulting in a higher overall mean food consumption for the premating phase (relative difference from controls: 15%). Females at 7500 ppm consumed less food than controls on the first treatment day. Thereafter, their food consumption generally remained close to control values (for one cage of the 7500 ppm females (cage no. 18) food consumption values were higher compared to controls on a few days during the pre-mating period). Overall, food consumption was considered not to be affected by treatment since no consistent differences from controls were noted. The statistically significant differences in females at 2000 and 7500 ppm noted on a single occasion during the post-coitum period (Days 22-23 when group means were based on only a few females) and the lactation period (Days 9-10) and for 7500 ppm the food consumption corrected for body weight in the lactation period (Days 12-13) were considered to be chance findings.
The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table in "any other information"

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters (red and white blood cells) were considered not to have been affected by treatment.
Isolated statistically significant variations noted in red blood cell parameters (slightly lower hemoglobin and hematocrit in females at 500 and 7500 ppm) were considered unrelated to treatment due to the absence of a dose-related trend.
Coagulation parameters were considered not to have been affected by treatment.
An isolated statistically significant variation noted in prothrombin time (lower in males at 2000 ppm) was considered unrelated to treatment due to the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few statistically significant differences were noted between the 7500 ppm group and the control group:
• Higher potassium in males (1.09 x of control). Values in 7500 ppm males and in one control male were at or slightly above the upper limit of the historical control range. (Historical control data period 2015-June 2017; potassium in male rats (mmol/L) mean = 3.94, P5-P95 = 3.54-4.37 (n=219))
• Lower total protein in females (0.92 x of control). Values in 7500 ppm females remained within the historical control range. (Historical control data period 2015-June 2017; total protein in female rats (g/L) mean = 61.8, P5-P95 = 54.7-68.1 (n=186))
In females a large variety in bile acids were noted, both in the control group and 7500 ppm females this is caused by an individual animal with a markedly higher value compared to the remainder of the group.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. In the absence of a dose-related response, the statistically significantly lower fore limb grip strength in females at 500 ppm was considered unrelated to treatment.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in organ weights.
Thyroid weight for 2000 ppm females was statistically significant decreased compared to controls; this was considered unrelated to treatment due to the absence of a dose-related trend.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 717 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

overall mean test item intake

3

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

overall mean test item intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Diet analyses

Accuracy: No test item was detected in the control diet.

The concentrations analyzed in the 500, 2000 and 7500 ppm diets were in agreement with target concentrations, with mean accuracies between 97% and 100%).

Homogeneity: The 500 ppm and 7500 ppm diets were homogeneous (coefficient of variation 2.9 and 5.0%, respectively).

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the text table below.

		Mean over means intake[mg test item/kg body weight/day] (mean range indicated between brackets)
sex	Study period	500 ppm	2000 ppm	7500 ppm
Males	Pre-mating	45 (41-51)	197 (168-238)	790 (623-1062)
	Post-mating	35 (31-47)	150 (134-238)	590 (500-819)
	Mean of means^a	41	181	717
Females	Pre-mating	42 (36-59)	177 (145-238)	737 (471-1035)
	Post-coitum	64 (44-76)	235 (116-296)	995 (648-1217)
	Lactation	113 (73-144)	472 (305-593)	1907 (1116-2514)
	Mean of means^a	71	281	1175

aMean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14x mean pre-mating) + (13x mean post-mating)) / 27

Females (Group 2 and 3): ((14 x mean pre-mating) + (26 x mean post-coitum) + (14 x mean lactation)) / 54

Females (Group 4): ((14 x mean pre-mating) + (23 x mean post-coitum) + (14 x mean lactation)) / 51

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of at least 7500 ppm was established, which corresponds to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively. These values are very close to the limit dose of 1000 mg/kg bw and therefore the final conclusion is that 'no adverse effects are observed'.

Executive summary:

Introduction: The objectives of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) were to determine the potential toxic effects of the substance when given via diet for a minimum of 28 days to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) no-observed-adverse-effect levels (NOAELs) were evaluated. In this section only the repeated dose parameters are presented. The fertility and developmental effects are presented in the reproductive toxicity sections.

Method: The dose levels in this study were based on the results of a dose range finder. Rats (10/sex) received food with vehicle or test substance at levels of 500, 2000 or 7500 ppm (nominal 30, 130 and 500 mg/kg bw/day, respectively).

Results: Accuracy and homogeneity of diet preparations were demonstrated by analyses. The intake of the substance was higher than expected and resulted in an overall average for the high dose of 717 mg/kg bw for males and 1175 mg/kg bw for females.

Clinical signs: Exposure to the test item up to 7500 ppm was well tolerated as indicated by the absence of adverse changes in the parental parameters examined in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations).

Body weight gainof females at 7500 ppm was somewhat reduced during lactation. As the associated decreases in mean body weight did not exceed 10% this finding was considered not to be adverse.

Heamatology: No adverse effect seen.

Clinical biochemistry: No adverse effects seen. A higher mean plasma level of potassium was noted in males treated at 7500 ppm. This change was regarded as non-adverse based on its modest magnitude (9% difference from controls) and the absence of changes indicative of target organ toxicity or alterations in the general function of the animals. A lower mean plasma level of total protein was observed in females treated at 7500 ppm. This change was regarded as non-adverse due to its slight magnitude (8%, within the historical control range, see results section) and the absence of any evidence of conditions known to be associated with decreases in total protein.

Organ effects: Absolute and relative weights, macroscopically and microscopically there were no adverse effect seen.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, a parental no-observed-adverse-effect level

(NOAEL) of at least 7500 ppm was established (corresponding to an overall mean test item intake of at least 717 and 1175 mg/kg bw/day for males and females, respectively).