Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data are available.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS for Extended one generation reproductive toxicity study (EOGRTS).

Substance name: 2,2'-[OXYBIS(METHYLENE)]BIS[2-ETHYLPROPANE-1,3-DIOL]

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- No existing GLP studies study available for this endpoint.
- No existing non-GLP studies study available for this endpoint.
- No historical human data available for this endpoint.
- No existing (Q)SAR information can properly assess this endpoint.
- No existing in vitro method could address this endpoint.
- No sufficient information available that could fulfill the standard information requirement through the weight of evidence approach.
- No information from structurally similar substance can be used to fulfill the standard information requirement through grouping and read-across.


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Extended one generation reproductive toxicity studies (EOGRTS) basic test design is the standard information requirement for substance registered for 1000 tonnes or more per year (Annex X, Section 8.7.3., column 1, of the REACH Regulation).


FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:

No evidence of effect on the reproductive organs was seen in a 28-day and a 90-day repeated dose toxicity study at dose levels up to 1000 mg/kg bw/d. An extended one generation reproductive toxicity study according to OECD 443 guideline, basic test design (cohorts 1A and 1B without extension to include a F2 generation) is proposed at the present time. The detailed study design of EOGRTS will be further assessed to ensure its adequate assessment of possible effects and avoid unnecessary vertebrate animal use.
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: to be confirmed.
- Exclusion of extension of Cohort 1B: No information indicate the genotoxicity, endocrine disruption related effect of this substance. No information indicate the substance leads to significant exposure or specific metabolism pattern that would require extended exposure. Therefore, extension of cohort 1B to include the F2 generation is not proposed.
- Exclusion of developmental neurotoxicity Cohorts 2A/2B and Cohort 3: No indication of the developmental neurotoxicity or immunotoxicity of this substance was seen from existing information. Cohort 2A/2B and cohort 3 are considered not needed at this moment.
- Route of administration: Oral
- Other considerations: A prenatal developmental toxicity study in rabbits in ongoing. The detail study design of EOGRTS will be further assessed once the results from the prenatal study are available to ensure its adequate assessment of possible effects and avoid unnecessary vertebrate animal use.

Based on above considerations, an extended one generation reproductive toxicity study according to OECD 443 guideline, basic test design (cohorts 1A and 1B without extension to include a F2 generation) is proposed at the present time. Further study design details will be provided when the ongoing OECD 414 study results are available.
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley SD rad is the species and strain of choice because there is ample experience and background data on this species and strain. It is also the species and strain used in other toxicity studies of this substance (ex. 90 days repeated dose study) which provide better results-comparison between studies.
Sex:
male/female
Route of administration:
oral: unspecified
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Extended one-generation reproductive toxicity study (EOGRTS)

No evidence of an effect on the reproductive organs was seen in a 28-day and a 90-day repeated dose toxicity study at dose levels of up to and including 1000 mg/kg bw/d. The absence of histopathological changes in reproductive organs in repeated dose toxicity study indicate that the substance will not have any adverse effects on fertility. An extended one generation reproductive toxicity study according to OECD 443 guideline, basic test design (cohorts 1A and 1B without extension to include a F2 generation) is proposed at the present time. The detailed study design of EOGRTS will be further assessed to ensure its adequate assessment of possible effects and avoid unnecessary vertebrate animal use.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity studies in rats as well as rabbits (both performed according to OECD test guideline 414) are available for Di-TMP.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 July to 06 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study with no deviations
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, UK
- Age at study initiation: 9 weeks old
- Weight at study initiation: 177-269 g on arrival; 197-307 g at the initiation of dosing
- Housing: 2 per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings, and enrichment (devices for hiding in and an object for chewing)
- Diet (e.g. ad libitum): SDS VRF-1 breeder diet, ad libitum
- Water (e.g. ad libitum): water from the public supply, ad libitum
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%): 42-62%
- Air changes (per hr): ten or more with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour cycle

IN-LIFE DATES: From: 27 July 2015 To: 12 August 2015
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose volume was 4 mL/kg, and the concentration of the test substance in the vehicle was 0, 25, 75 and 250 mg/mL. Dose volumes were based on the most recent body weight recorded for each animal. The control item, corn oil, was aliquoted weekly, stored in a refrigerator set to maintain 4°C and dispensed daily. The prepared control item was removed from the refrigerator and stirred for at least 30 minutes prior to dosing, and continuously during dosing. Prior to formulation, the test item was ground in a mortar, since it was supplied as flakes. The required amount of ground test item was weighed into a pre-labelled container according to instructions from the formulation computerised system (Dispense 8). The appropriate amount of vehicle was then added to the container and the formulation was magnetically stirred until visibly homogenous. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing and continuously during dosing.

Stability analyses performed previously in conjunction with Charles River Study No. 434348 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study for at least 8 days at 2-8°C, in the dark.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by Gas Chromatography using a validated analytical procedure. Duplicate top, middle and bottom samples (middle only for control) of 0.1 mL for each sampling time point were collected and sent to the analytical laboratory for analysis. Triplicate sets of top, middle and bottom samples (middle only for control) of the same volume were also collected and retained as backup samples. Concentration results were considered acceptable if mean sample concentration results were within 10% of theoretical concentration and individual sample concentrations were within 15% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤10%.
Details on mating procedure:
Not applicable - time mated females were obtained direct from the supplier.
Duration of treatment / exposure:
From Days 6-19 of gestation
Frequency of treatment:
Once daily
Duration of test:
Day 0 (mating) to Day 20 of gestation (scheduled termination)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen by the Sponsor following review of data from a 28 day repeated dose study of DiTMP in rats. On that study, tubular basophilia and inflammatory cell infiltration in the kidney were noted in males at all dose levels (40, 200 or 1000 mg/kg/day); however, these findings did not follow a clear dose-related pattern, were of little toxicological significance and were not noted in females. The NOAEL was therefore considered to be 1000 mg/kg bw/d.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were observed twice daily, once in the morning and once towards the end of the working day throughout the study for general health/mortality and moribundity. Animals were observed prior to dosing and regularly throughout the day for reaction to treatment on each day of dosing. The onset, intensity and duration of these any signs were recorded, with particular attention paid to the first hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
Animals were given a detailed examination once pretreatment and daily from Day 6 of gestation.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 4 of gestation and daily from Days 6 to 20 of gestation.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured daily throughout the study, beginning on Day 4 of gestation (first measured quantity given on Day 3 of gestation).

WATER CONSUMPTION: Yes
Water consumption was monitored by visual inspection of the water bottles only. As no intergroup differences were noted, consumption was not measured by weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All adult animals were given an external examination, and then the contents of the cranial, thoracic and abdominal cavities were examined macroscopically, with representative samples of abnormal tissues being fixed in neutral buffered 10% formalin. The reproductive tract was dissected out and examined
Ovaries and uterine content:
The reproductive tract was dissected from the abdominal cavity. The gravid uterus weight was recorded. The ovaries and uterus were examined for number and distribution of: Corpora lutea; Implantation sites; Placentae (size, colour or shape) – only abnormalities were recorded; Live and Dead Fetuses; Early and Late Resorptions
Fetal examinations:
External abnormalities - Each fetus was examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no signs of maceration), or a late embryonic death (macerated tissue identifiable as an embryofetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may have been of varying size).

Visceral examinations and sex - Half of the viable foetuses from each uterus were fixed in methylated ethyl alcohol, examined internally for sex and eviscerated following fixation, the viscera were not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouins’ fluid. The foetuses fixed in Bouins’ fluid were examined for soft tissue abnormalities and sex by a freehand sectioning technique derived from Wilson.

Skeletal examination - The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.
Statistics:
Means and standard deviations were calculated for body weight, food consumption and selected pregnancy data. Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to relevant classification variables (e.g., sex, occasion). Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) are reported whenever possible. Inferential statistics were performed for body weight, food consumption and body weight change when possible, but excluded semi-quantitative data and any group with less than 3 observations.

ANOVA - Levene’s test was used to assess the homogeneity of group variance parametric assumption at the 5% significance level. Datasets with at least three groups was compared using an overall one-way ANOVA F-test or Kruskal-Wallis test (if parametric assumptions were not met) at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Dunnett’s or Dunn’s test, respectively, if the overall test is significant. Datasets with two groups were compared using a two-sided t-test or Wilcoxon Rank-Sum test, respectively. All significant pairwise comparisons were reported at the 0.1, 1 and 5% significance levels.
Indices:
Not applicable
Historical control data:
Available at the test facility
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no mortalities during the study. An increased incidence of ploughing behaviour (animal burrowing through bedding with its head) was noted in all treated groups; this observation was absent in Controls. This finding was transient, being noted immediately post dosing, and was no longer evident 1 hour after dosing, and was generally noted on 2-3 occasions between Days 14-20 of gestation in affected animals. All other clinical observations were considered to be incidental background findings commonly observed in this species and unrelated to treatment with DiTMP.
There were no effects on group mean body weights, group mean food consumption, and there were no abnormal findings detected at necropsy.

Pregnancy performance parameters and fetal weights were similar between all groups. Slight intergroup differences in embryonic deaths and fetal weights were noted between groups; however, these were considered to be incidental as they were within normal variation and were too small to be attributed to treatment with DiTMP.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: In all treated groups, dose-related increased incidences of misshapen scapula(e) were noted when compared with Controls

Details on embryotoxic / teratogenic effects:
In all treated groups, dose-related increased incidences of misshapen scapula(e) were noted when compared with Controls (14 fetuses in 10 litters at 100 mg/kg/day, 17 fetuses in 9 litters at 300 mg/kg/day and 23 fetuses in 10 litters at 1000 mg/kg/day, compared with 6 fetuses in 4 Control litters).
At 1000 mg/kg/day, increased incidences of unossified hyoid bone and 5th metacarpal(s) were noted when compared to Controls. Decreased incidences of ossified anterior atlas, cervical vertebral centra, phalangeal elements and sacrocaudal vertebrae with connections between centrum and arches were also observed in this group, when compared with Controls.
Renal pelvic dilation, which was noted in a small number of fetuses and litters at 1000 mg/kg/day, was considered unlikely to be related to treatment due to the low incidence and since the number of fetuses and litters affected is within background control ranges at the Test Facility.
The type and distribution of major fetal abnormalities and all other minor fetal abnormalities and variants and skeletal ossification parameters did not indicate any association with treatment. Slight intergroup differences were considered to be incidental and unrelated to treatment with DiTMP.
Dose descriptor:
NOAEL
Effect level:
< 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
skeletal malformations
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
skeletal malformations
Abnormalities:
not specified
Developmental effects observed:
not specified

Dose formulation analyses: Analysed Group 2, 3 and 4 formulations from Study Weeks 1 and 2 were found to be within the concentration acceptance criteria of ±10% of theoretical concentration (actual mean concentration range -7% to +1.7% of theoretical concentration). A low relative standard deviations of ≤4.5% also indicated that all formulations were homogenous. The absence of DiTMP from analysed control formulations was also confirmed.

Summary of Group Incidences of Treatment Related Clinical Observations

 

Dose Group/Dose Level (mg/kg bw/d)

Observation

1 (0)

2 (100)

3 (300)

4 (1000)

Ploughing behaviour

0/24

11/24

14/24

24/24

Group Incidences of Minor Fetal Abnormalities and Variants

Abnormality/Variant

Group/Dose Level (mg/kg bw/d)

1

(0)

2 (100)

3 (300)

4 (1000)

Incidence of Fetuses (Litters)

Skeletal

Basisphenoid incompletely ossified, bilateral jugal misshapen

0

0

1(1)

0

Basioccipital misshapen

0

0

0

1(1)

Cranial bone(s) discrete unossified/incompletely ossified area(s)

3(2)

0

1(1)

2(1)

Cervical vertebral arch(es) increased ossification

11(7)

5(4)

12(8)

8(7)

Scapula(e) misshapen

6(4)

14(10)

17(9)

23(10)

Bilateral humerus incompletely ossified

0

0

0

1(1)

Sternebra bifurcated, xiphoid cartilage incomplete

0

1(1)

0

0

Rib(s) minimally kinked

0

1(1)

1(1)

2(2)

Rib(s) incompletely ossified

0

0

0

1(1)

Additional ossified area arising from sternebra

2(2)

0

1(1)

1(1)

Rib(s) costal cartilages asymmetrically aligned

7(7)

7(6)

6(6)

1(1)

Rib(s) costal cartilage(s) not attached to sternum

4(3)

2(1)

2(2)

1(1)

Pelvic girdle cranial displacement: Unilateral

1(1)

0

0

0

Pelvic girdle caudal displacement

 

 

 

 

                                               Unilateral:

0

0

1(1)

0

                                               Bilateral:

0

1(1)

0

0

Number with minor abnormality/variant

31(18)

29(15)

37(19)

36(16)

Total number examined skeletally

164(24)

150(24)

157(24)

161(24)

Number of ribs

13th vestigial rib(s)

4(3)

2(2)

0

1(1)

13th reduced rib(s)

2(2)

0

1(1)

3(1)

13 complete rib(s)

149(24)

142(24)

152(24)

155(24)

Vestigial supernumerary rib(s) on 1st lumbar vertebra

9(6)

6(5)

3(3)

2(2)

Reduced supernumerary rib(s) on 1st lumbar vertebra

0

0

1(1)

0

Group Incidences of Skeletal Ossification Parameters

Parameter

Group/Dose Level (mg/kg bw/d)

1 (0)

2 (100)

3 (300)

4 (1000)

Incidence of Fetuses (Litters)

Incomplete ossification affecting:

4 skull bones

6(4)

2(2)

5(3)

7(5)

3 skull bones

26(14)

14(7)

16(9)

21(12)

Cervical vertebral arch(es)

1(1)

1(1)

1(1)

0

Thoracic vertebral centrum(a)

9(7)

12(9)

8(6)

8(7)

Lumbar vertebral arch(es)

0

0

1(1)

0

Lumbar vertebral centrum(a)

0

0

1(1)

0

Pubis/es

5(3)

5(4)

1(1)

2(2)

Ischium/a

1(1)

0

3(2)

0

Sacral vertebral arch(es)

18(3)

6(5)

6(3)

8(6)

2nd and/or 4th metacarpal(s)

5(4)

1(1)

3(3)

1(1)

Unossified:

Hyoid

13(9)

4(4)

8(4)

20(12)

5th metacarpal(s)

29(13)

20(10)

29(14)

41(13)

5th metatarsal(s)

0

1(1)

1(1)

0

Ossified:

Anterior arch of atlas

51(17)

51(20)

41(13)

36(17)

>2 cervical vertebral centra

15(11)

13(8)

17(8)

10(8)

One or more sacrocaudal vertebrae with connection

between centrum and arch(es)

46(19)

46(21)

53(17)

35(16)

Phalangeal elements

23(9)

33(12)

26(11)

13(8)

Mean number of caudal vertebral centra

4.1

4.0

4.1

3.8

Number of sternebrae retarded:

                                               0

69(24)

71(22)

85(21)

60(19)

                                               1

75(22)

58(21)

59(21)

70(22)

                                               2

18(11)

12(11)

11(9)

27(14)

                                               >2

2(2)

9(4)

2(2)

4(4)

Total number examined skeletally

164(24)

150(24)

157(24)

161(24)

Conclusions:
The NOAEL for maternal toxicity was considered to be 1000 mg/kg bw/d, however a developmental NOAEL could not be established.
Executive summary:

The potential for DiTMP to cause prenatal developmental toxicity was investigated in pregnant Sprague-Dawley rats, according to OECD 414. DiTMP was administered to groups of 24 time-mated rats by gavage at doses of 0, 100, 300 or 1000 mg/kg bw/d in corn oil. Animals were dosed over Days 6-19, inclusive, of gestation (where the day of detection of mating was Day 0 of gestation) and regularly checked for clinical signs of toxicity, body weight and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and embryofetal development.

Dosing with DiTMP was not associated with any maternal body weight or food consumption effects or gross necropsy findings. A dose-related increased incidence of ploughing behaviour was noted in all treated groups when compared to controls; however, as this findings was transient, noted only on a few occasions in each individual and is thought to be a reaction to unpleasant taste of dosing formulations, it was considered not to be adverse at any level.

Pregnancy performance and fetal weights in all groups were similar, and there were no major fetal abnormalities which were considered to be related to treatment with DiTMP. In all treated groups, a dose-related increased incidence of misshapen scapula(e) was noted when compared with controls. This finding is difficult to interpret as it had only recently been noted at the Test Facility in a small number of studies at low incidence, therefore background control data on this finding is limited, and the incidences in all treated groups on this study are above the background range. The significance of this finding could therefore not be determined. At 1000 mg/kg/day, a general trend toward delayed ossification was noted (which included increased incidences of unossified hyoid bones and 5th metacarpals and decreased incidences of ossified anterior atlas, cervical vertebral centra, phalangeal elements and sacrocaudal vertebrae with connections between centrum and arches) when compared with Controls. It was therefore concluded from the results of this study, that the maternal NOAEL was 1000 mg/kg bw/d. A developmental NOAEL cannot be established due to an increased and dose-related incidence of misshapen scapulae at all dose levels and a trend towards delayed ossification at 1000 mg/kg bw/d.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-18 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Name of test material (as cited in study report): Di-Trimethylolpropane
- Chemical name (IUPAC): 2,2’-[Oxybis(methylene)]bis[2-ethylpropane-1,3-diol]
- Analytical purity: 99%
- Lot/batch No.: 170703932
- Expiration date of the lot/batch: 13 July 2019
- Storage condition of test material: Room temperature
Species:
rabbit
Strain:
New Zealand White
Remarks:
specific-pathogen-free
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia S.r.l.
- Age at study initiation: 13 weeks of age (females) and at least 25 weeks of age (males)
- Weight at study initiation: At least 2.5 kg body weight (females) and more than 3.5 kg body weight (males)
- Fasting period before study: not specified
- Housing: The rabbits were housed in a limited access animal facility. The animals were individually housed in grid-bottomed metal cages suspended over trays. Each cage tray held absorbent paper which was inspected and changed as necessary.
- Diet: A commercially available laboratory rabbit diet (2RB, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
- Water: ad libitum to each cage via water bottles
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C ± 2 °C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): The rooms were lit by artificial light for 12 hours each day.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
2% CMC in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of the test item was suspended in the vehicle (2% carboxymethyl cellulose in water) during stirring and heating up to approximately 60 °C. Once the test item was dissolved, the formulations were kept between 35-40 °C in water bath on magnetic stirrer. The formulations were prepared daily. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was composed by carboxymethyl cellulose 2% in water. Administrations of corn oil at dose volume of 2 mL/kg and above are not tolerated by pregnant rabbit (EPA Archived Document OPPTS 870.3770, 1996). The solubility test of the test item in corn oil at dose volume of 1 mL/kg indicated it was not soluble and no suspension was formed. Therefore, it was decided to select another vehicle in order to avoid toxicity on pregnant rabbits and the selected vehicle of carboxymethyl cellulose 2% in water gave a good suspension with the test item.
- Concentration in vehicle: 2% carboxymethyl cellulose in water
- Amount of vehicle (if gavage): 5 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in this study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (5.5 hour at +40°C). The range of tested concentrations was between 20 and 200 mg/mL. Acceptance criteria for the validation was linearity R > 0.98, accuracy 85-115% and precision CV<10%.

In addition samples of the formulations prepared during the study (the first and the last week of treatment) were analysed to check the homogeneity and concentration (acceptance criteria ± 15% of the theoretical value for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Analyst 1.6.2.
Details on mating procedure:
- Impregnation procedure: co-housed
- M/F ratio per cage: 1 male/1 female per cage (one female was placed into the home cage of the one male)
- Length of cohabitation: Each female remained with the male (in the home cage of the male) for at least 1 hour after successful mating was observed.
- Proof of pregnancy: Day of coitus referred to as day 0 of gestation
Duration of treatment / exposure:
Day 6 to day 28 post coitum
Frequency of treatment:
Once a day
Duration of test:
Females that completed the scheduled treatment period were euthanised on day 29 post coitum.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a previous preliminary study on embryo-foetal development in the rabbit (RTC no. Y0220, not GLP-compliant).
The outcome of this preliminary study indicated that the treatment with Di-Trimethylolpropane at 1000 mg/kg/day caused maternal toxicity in terms of mortality and reduction in body weight. At this dose level, one female died, and the mean body weight was significantly reduced during the last week of treatment (7% reduction on Day 23 post-partum and 13% reduction on Day 26 post-partum).
No adverse changes were detected at 100 and 300 mg/kg/day. Based on the outcome of this study, the high dose level for the subsequent reproductive/developmental toxicity study should be lower than 1000 mg/kg/day.
Therefore, the dose levels of 100, 300 and 750 mg/kg/day were selected for the subsequent prenatal developmental toxicity study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during treatment at the same time interval each day
- Cage side observations checked include abrasions/injuries on the body, hair loss, detection of faecal matters and staining on cage tray.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal and group (as a mean) determined: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 (post coitum)
- Organs examined: Ovaries and uteriCAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once daily during treatment at the same time interval each day
- Cage side observations checked include abrasions/injuries on the body, hair loss, detection of faecal matters and staining on cage tray.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group (Day 0 post coitum) and on days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 post coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal and group (as a mean) determined: Yes
- Time schedule for examinations: Food consumption was recorded on Days 3, 6, 9, 12, 15, 18, 21, 23, 26 and 29 of post coitum starting from Day 0 post coitum.

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 (post coitum)
- Organs examined: Ovaries and uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Gross evaluation of placentae
Fetal examinations:
- Examination of number, sex and weight of all living foetuses: yes
- Examination of number and sex of dead foetuses: yes
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes: [half of all living foetuses, i.e. every other second living foetus]
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data.

Statistical analyses of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams’ test.
Indices:
Group mean values for body weight and food consumption of pregnant females, terminal body weight, gravid uterus weight, absolute weight gain (terminal body weight minus gravid uterus weight minus body weight at Day 0 post coitum), litter size, intra-uterine deaths, corpora lutea count, total implantation loss and pre- and post-implantation loss were calculated. Data from non-pregnant animals were not included in group mean calculations. Animals which aborted were included in body weight calculations up to the day of abortion.

Litter data: Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss% = (no. of corpora lutea – no. of implantations)/(no. of corpora lutea) x 100

Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss% = (no. of implantations – no. of live foetuses)/(no. of implantations) x 100

Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss% = (no. of corpra lutea – no. no. of live fetuses)/(no. of corpora lutea) x 100

Sex ratios of the foetuses were calculated as the percentage of males.
The number of foetuses affected with structural deviations and the corresponding litter
percentage were calculated. All derived values (e.g. means, percentages, ratios) were first
calculated within the litter and the group values derived as a mean and standard deviation
of individual litter values. Foetal structural deviations were expressed as the percentage
of affected foetuses relative to all foetuses examined per group, as well as in terms of the
mean litter percentage of affected litters.
Historical control data:
no data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hair loss was recorded in a few animals (no more than 2 per dose group) in all dose groups. Reduced and soft faeces were seen in medium- and high-dose groups with a similar incidence (up to 2 animals). Red staining and foetuses in cage tray were recorded in the 2 females that were humanely sacrificed before end of study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Unscheduled deaths occurred during the study. Two females, one in the medium dose group and one in the high dose group, were humanely sacrificed for abortion on days 25 and 23 post coitum, respectively.
Fate: The total number of females at term was 20 in the control group, 18 in the low and high dose groups, and 19 in the mid-dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease of terminal body weight (5% decrease) recorded at the end of the study was observed in the high-dose females receiving 750 mg/kg/day (4016.11 g) when compared to controls (4240.50 g). No significant changes were detected in body weight between control and the low- or medium-dose females.

Statistically significant reduction in absolute body weight gain was recorded in the high-dose females receiving 750 mg/kg/day starting from Day 12 post coitum up to the end of the study. At the end of the study (i.e. day 29 post coitum), there was a 39.9% decrease in the mean absolute body weight gain of the high-dose females (318.89 g) compared to that of the control females (530.50 g).

See Tables 1 and 2 in box „Any other information on results incl. tables”.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a consistent trend of reduced food consumption in the high dose females receiving 750 mg/kg/day starting from Day 9 post coitum. On day 29 post coitum, there was a 15.6% decrease (with statistical significance) in the mean food consumption of the high-dose females (141.38 g) compared to that of the control females (167.51 g).

See Table 3 in box "Any other information on results incl. tables" for results.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was slight decrease in uterus weight in all treated groups compared to controls. However, no dose-response relationship or statistical significance in the decrease was observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted during the macroscopic observation of the females. The sporadic changes such as hair loss in two high-dose females or abrasion in one high-dose female were not attributable to treatment.

Of the two females that were humanely sacrificed before end of study, no macroscopic findings were seen in the high-dose female, whereas moderate hair loss of the abdominal region was seen in the medium-dose female.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Slight maternal toxicity was present in the high dose females receiving 750 mg/kg/day as demonstrated by a statistically significant reduction of absolute body weight gain, food consumption and terminal body weight. No significant differences in these parameters were detected in the low- and medium-dose females when compared to controls.

At necropsy all animals at term did not show abnormalities that could be considered related to treatment.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Two females (one in the medium-dose group and one in the high-dose group) were humanely sacrificed on days 25 and 23 post coitum, respectively, due to abortions. This is considered not to be treatment-related since this low incidence of abortion is within the range of the historical control data.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.
Dead fetuses:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals. See Table 4 in box "Any other information on results incl. tables" for results.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Two los dose females and one high dose female were found not pregnant at final sacrifice.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no difference in the mean foetal weight of both sexes, but there was a significant decrease in male foetal weight at the highest dose.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant difference observed between treated and control animals.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No abnormalities were detected in all groups (treated and control).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were recorded.

Malformation recorded in one foetus of the control group (lumbar hemivertebrae) and in two different foetuses of the low-dose group (articulation point absent in the pelvic girdle and thoracic hemivertebrae) were considered spontaneous in origin.
Visceral malformations:
no effects observed
Description (incidence and severity):
No abnormalities were detected in all groups (treated and control).
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The uterine examinations did not reveal any effects on foetuses on term. The mean foetal and litter weights of all treated groups were comparable to controls. At the external and internal examination of foetuses no treatment-related abnormalities were described. The examination of foetal fixed head and skeleton did not reveal any treatment-related findings.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
No treatment-related developmental effects were observed.
Key result
Developmental effects observed:
no
Treatment related:
no

Table 1: Summary of maternal terminal body weight group data

Group   Terminal body weight [g]
Control N 20
  Mean [g] 4240.50
  SD 229.04
Low dose N 18
  Mean [g] 4080.00
  SD 202.22
Medium dose N 19
  Mean [g] 4145.79
  SD 259.51
High dose N 18
  Mean [g] 4016.11*
  SD 254.40

N = number of animals; SD = standard deviation; * = mean value of group is significantly different from control (Statistical analysis: Kruskall Wallis test and William’s test if group differences are different from control at p < 0.05)

Table 2: Summary of the maternal absolute body weight gain group data

    Post coitum                
Group   Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21 Day 23 Day 26 Day 29
Control N 20 20 20 20 20 20 20 20 20 20
  Mean [g] 0.00 83.00 139.00 195.50 310.50 322.50 347.50 380.00 450.00 530.50
  SD 0.000 39.484 42.785 67.938 121.373 111.868 122.426 125.865 131.869 134.926
Low dose N 18 18 18 18 18 18 18 18 18 18
  Mean [g] 0.00 75.56 117.22 167.78 233.33*C 265.56 289.44 304.44 347.78 383.33*D
  SD 0.000 49.967 40.410 60.348 87.515 72.048 90.064 97.088 126.749 150.646
Medium dose N 20 20 20 20 20 20 20 20 19 19
  Mean [g] 0.00 67.00 109.50 151.50 222.00 234.50 255.00 278.00 355.79 416.84
  SD 0.000 60.966 69.546 75.413 160.348 198.321 237.985 241.739 142.568 140.121
High dose N 19 19 19 19 19 19 19 19 18 18
  Mean [g] 0.00 73.68 68.42+C 82.11+C 127.37+C 164.74*C 162.11*C 162.11+C 267.22+D 318.89+D
  SD 0.000 34.835 66.855 144.936 225.606 251.868 264.902 272.918 133.320 201.111

N = number of animals; SD = standard deviation; *D Dunnett LSD Test Significant at the 0.05 level; +D Dunnett LSD Test Significant at the 0.01 level; *C Cochran and Cox Test Significant at the 0.05 level; #C Cochran and Cox Test Significant at the 0.001 level

Table 3: Summary of food consumption group data

    Post coitum                
Group   Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21 Day 23 Day 26 Day 29
Control N 20 20 20 20 20 20 20 20 20 20
  Mean [g] 186.84 205.52 212.62 204.35 186.08 209.81 206.60 201.95 172.37 167.51
  SD 25.954 15.581 18.251 21.461 31.237 25.565 23.504 36.350 29.793 31.199
Low dose N 18 18 18 18 18 18 18 18 18 18
  Mean [g] 185.54 216.10 212.42 202.87 184.09 210.55 218.25 227.77 177.28 191.37
  SD 18.290 28.044 16.907 17.372 30.567 31.961 31.958 52.702 38.200 34.313
Medium dose N 20 20 20 20 20 20 20 20 19 19
  Mean [g] 191.47 215.08 201.93 193.47 174.00 187.02 192.96 179.37 153.29 154.17
  SD 26.979 25.751 29.414 26.430 43.247 43.034 45.757 67.851 31.852 33.764
High dose N 19 19 19 19 19 19 19 19 18 18
  Mean [g] 187.52 206.44 180.14#C 168.43+C 136.54+D 166.51+D 175.91*C 166.73 148.28 141.38*D
  SD 20.933 18.452 29.868 41.955 35.970 45.617 54.445 59.560 27.136 32.587

N = number of animals; SD = standard deviation; *D Dunnett LSD Test Significant at the 0.05 level; +D Dunnett LSD Test Significant at the 0.01 level; *C Cochran and Cox Test Significant at the 0.05 level; #C Cochran and Cox Test Significant at the 0.001 level

Table 4: Summary of reproductive/developmental endpoint group data

Group

 

Corpora Lutea

Implantation

Pre-Imp. Loss

Post-Imp. Loss

Early Resorption

Late Resorption

Male Sex Percent

Dead foetuses

Live foetuses

Male foetal weight mean [g]

Female foetal weight mean [g]

Litter weight [g]

Control

Mean

9.60

9.30

0.30

4.03

0.00

0.25

50.10

0.00

9.05

45.35

43.10

390.81

 

SD

2.21

2.49

0.57

8.09

0.00

0.79

15.94

0.00

2.39

6.11

5.61

81.10

 

N

20

20

20

20

20

20

20

20

2039

20

20

20

Low dose

Mean

8.72

8.00

0.72

9.87

0.11

0.06

44.17

0.00

7.83

43.96

43.44

332.11

 

SD

2.08

2.57

1.02

14.75

0.32

0.24

21.63

0.00

2.46

5.35

6.05

91.32

 

N

18

18

18

18

18

18

18

18

18

18

18

18

Medium dose

Mean

9.89

9.68

0.21

2.86

0.11

0.26

48.16

0.00

9.32

44.03

43.11

392.62

 

SD

2.11

2.26

0.42

6.50

0.32

0.93

17.11

0.00

2.40

7.91

7.16

77.63

 

N

19

19

19

19

19

19

19

19

19

19

19

19

High dose

Mean

9.50

9.28

1.11

5.49

0.06

0.00

48.89

0.00

9.22

40.35*

40.28

370.84

 

SD

1.86

1.27

2.32

7.24

0.24

0.00

16.33

0.00

1.35

3.37

3.63

48.22

 

N

18

18

18

18

18

18

18

18

18

18

18

18

N = number of animals or litters (depending on endpoint); SD = standard deviation; * = mean value of group is significantly different from control (Statistical analysis: Kruskall Wallis test and William’s test if group differences are different from control at p < 0.05)

Conclusions:
In a prenatal developmental toxicity study performed in accordance with OECD 414, slight maternal toxicity was observed in rabbits at the highest tested dose of 750 mg/kg/day without any indication of developmental effects. Hence, 300 mg/kg/day is determined as the NOAEL for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.
Executive summary:

In a prenatal developmental toxicity study performed in accordance with OECD 414, di-trimethylolpropane (99% purity) was administered to New Zealand White pregnant rabbits (20 females per dose) in 2% carboxymethyl cellulose in water at dose levels of 0, 100, 300 or 750 mg/kg/day from day 6 to day 28 post coitum. Control animals received 2% carboxymethyl cellulose in water as vehicle at the same treatment period as the test item-treated animals. Animals were sacrificed on day 29 post coitum and subjected to post mortem examination. The females treated with 750 mg/kg/day exhibited slight toxicity as indicated by the reduction in body weight, absolute body weight gain and food consumption when compared with control females. No significant differences of the aforementioned parameters were observed in the females treated with 100 or 300 mg/kg/day. At necropsy, macroscopic examination of the females revealed no treatment-related changes.

 

The uterine examinations did not reveal any effects on the foetuses. The mean foetal and litter weights of all treated groups were comparable to controls. At the external and internal examination of foetuses no treatment-related abnormalities were described. The examination of foetal fixed head and skeleton did not reveal any treatment-related findings.

 

Based on the results from this study, 300 mg/kg/day is determined as the NOAEL (No-Observed-Adverse-Effect-Level) for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two GLP- and OECD test guideline-compliant studies are available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Prenatal developmental toxicity study with Di-TMP in rats

In a prenatal developmental toxicity study performed in accordance with OECD 414, di-trimethylolpropane (99% purity) was administered to New Zealand White pregnant rabbits (20 females per dose) in 2% carboxymethyl cellulose in water at dose levels of 0, 100, 300 or 750 mg/kg/day from day 6 to day 28 post coitum. Control animals received 2% carboxymethyl cellulose in water as vehicle at the same treatment period as the test item-treated animals. Animals were sacrificed on day 29 post coitum and subjected to post mortem examination. The females treated with 750 mg/kg/day exhibited slight toxicity as indicated by the reduction in body weight, absolute body weight gain and food consumption when compared with control females. No significant differences of the aforementioned parameters were observed in the females treated with 100 or 300 mg/kg/day. At necropsy, macroscopic examination of the females revealed no treatment-related changes.

 

The uterine examinations did not reveal any effects on the foetuses. The mean foetal and litter weights of all treated groups were comparable to controls. At the external and internal examination of foetuses no treatment-related abnormalities were described. The examination of foetal fixed head and skeleton did not reveal any treatment-related findings. Based on the results from this study, 300 mg/kg/day is determined as the NOAEL (No-Observed-Adverse-Effect-Level) for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

 

Justification for selection of Effect on developmental toxicity: via oral route: In the rats, a dose-related increased incidence of misshapen scapula(e) was noted when compared with controls. This finding is difficult to interpret as it had only recently been noted at the Test Facility in a small number of studies at low incidence; background control data on this finding is limited, and the incidences in all treated groups on this study are above the background range. It is therefore concluded from the results of this study that the maternal NOAEL was 1000 mg/kg bw/d and the developmental LOAEL was <100 mg/kg bw/day. Further data from the Test Facility on the background incidence of this finding from ongoing and future studies will enable the toxicological significance of the finding (variation) to be put in context.

 

On the other hand, in the rabbits, a prenatal developmental toxicity study, slight maternal toxicity was observed in rabbits at the highest tested dose of 750 mg/kg/day without any indication of developmental effects. Hence, 300 mg/kg/day is determined as the NOAEL for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

Toxicity to reproduction: other studies

Description of key information

No other studies.

Justification for classification or non-classification

The results of the 28-day repeated dose toxicity study do not indicate the reproductive system as a target of toxicity. A 90-day repeated dose toxicity study in the rat has also been conducted where no toxicologically relevant findings were noted.

The rat developmental toxicity study shows a dose-related increase in the incidence of a single foetal skeletal finding in all treated groups in the apparent absence of maternal toxicity. Interpretation of this finding (currently classed by the Test Facility as a minor abnormality) is difficult as it had only become apparent in controls in two studies prior to the Di-TMP study. Due to the increased background incidence of the finding, it is likely to be reclassified as a variation by the Test Facility. Furthermore, the relevance of the finding can only reliably be put in context when further background data from ongoing/future studies become available.

In a prenatal developmental toxicity rabbit study of Di-TMP performed in accordance with OECD 414, slight maternal toxicity was observed in rabbits at the highest tested dose of 750 mg/kg/day without any indication of developmental effects. Hence, 300 mg/kg/day is determined as the NOAEL for maternal toxicity and 750 mg/kg/day as the NOAEL for developmental toxicity.

Given the lack of adverse reproductive findings in the rat from repeated dose toxicity studies, the uncertainty of the data interpretation in the rat developmental toxicity study and the lack of developmental effects in the rabbits, no classification for the test item Di-TMP is therefore justified for reproductive toxicity under according to EC Regulation 1272/2008 taking a weight-of-evidence approach.