Amines, N-(C18-unsatd. alkyl)trimethylenedi-, ethoxylated

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November - 29 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tris(hydroxyethyl) oleyl diaminopropane

IUPAC Name:

Tris(hydroxyethyl) oleyl diaminopropane

Test material form:

other: Clear amber liquid

Details on test material:

- Name of test material (as cited in study report): Tris(hydroxyethyl) oleyl diaminopropane
- Substance type: Clear amber liquid
- Physical state: liquid
- Purity: 92.3 area% (Di amine + 3EO)
- Lot/batch No.: S001023
- Expiration date of the lot/batch: 09 July 2017
- Storage condition of test material: At room temperature in the dark under nitrogen
- Volatile: No
- Test substance handling: Flush container with nitrogen after handling
- Density: 940 kg/m3 at 20°C
- pH: 10 at 1% solution
- Stability in Propylene glycol: Stability for at least 6 hours at room temperature is confirmed over the concentration range 0.2 to 5 mg/mL

Test animals

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 295 gr (males) or 202 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 – 21.3°C
- Humidity (%): 37 - 68%
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Temporary deviations from the minimum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 06 November - 29 December 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for density of the test substance and specific gravity of the vehicle. No correction was made for purity of the test substance.
- Storage conditions of formulations: At ambient temperature under nitrogen.
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX and on information provided by the Sponsor.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Detection of mating was not confirmed for one female of Group 1, which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method (NOTOX Project 497194). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One female of Group 2 was not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1, 5, 25 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a Range Finding and MTD study (NOTOX Project 497209, see 7.5.1).
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily detailed clinical observations were made in all animals between 1 and 2 hours post-dose (based on results of the Range Finding and MTD study, NOTOX Project 497209). Once prior to start of treatment and at weekly intervals during the treatment period clinical observations
performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
Arena observations were conducted prior to dosing, instead of at 1-2 hours after dosing. The clinical signs recorded at 1-2 hours after dosing suggest that no abnormal findings would have been noted had the arena observations also been conducted at 1-2 hours after dosing. Based on the conducted clinical observations, an adequate evaluation of the study results could still be made.
On Days 1, 9 and 19 clinical signs were recorded later than between 1 and 2 hours after dosing, with a maximum of approximately 23 minutes. This deviation was of an incidental and slight nature. Based on the conducted clinical observations an adequate evaluation of the study results could still be made.

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

FOOD CONSUMPTION:
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: 5 animals/sex/group)
- Battery of functions tested: According to test guidelines

Oestrous cyclicity (parental animals):

Not determined.

Sperm parameters (parental animals):

Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group and animals suspected to be infertile).

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).
The last litter check was not recorded for two litters of Group 1 and one litter of group 4. The technician most likely examined all animals because the litters had to be handled when moving them to the necropsy room. Observations were conducted for these litters that same day as part of the external examination conducted before doing the macroscopic examination, and no findings were noted for any animal. In addition, no clinical signs were noted for any of these animals on the day prior to the last litter check.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY:
- All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY:
- According to test guidelines
From one male of Group 4, the left thyroid could not be found during weighing after fixation and hence no thyroid was weighed and the left thyroid was not available for histopathology. One eye of one male of Group 4 was not available for histopathology. Sufficient organ weight/histopathological data were available for evaluation.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Ref. 5 Wilcoxon, F. Individual comparisons by ranking methods. Biometrics, 1, 80-83 (1945).

Reproductive indices:

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

Offspring viability indices:

- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Details on results (P0)

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 25 mg/kg was sacrificed in extremis on Day 17. This animal showed wateryclear fluid in the thoracic cavity at necropsy. Microscopic findings consisted of a moderate subepicardial and atrial inflammation of the heart and slight to moderate pleural fibrosis with inflammation and alveolar fibrinous contents with a mononuclear inflammation of the lungs. No further mortality occurred among animals of this dose group, and none of the surviving animals showed any signs of imminent death/moribundity. Overall, these data strongly suggest that its death was gavage related.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted included scabs, alopecia, and swelling of the left hind paw. These findings 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. At the incidence observed, these were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. Motor activity was similar between the groups. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. The statistically significant lower body weight gain of males at 25 mg/kg over Days 1-8 of the premating period and over Days 8-15 of the mating period was slight in nature. No toxicological relevance was therefore ascribed to these changes.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
Females at 5 and 25 mg/kg showed a statistically significant higher relative and absolute neutrophil counts. Absolute white blood cell count (WBC) also showed a dose-related trend towards an increase (the mean at 25 mg/kg being outside the range considered normal), but did not achieve statistical significance. The statistically significant lower relative lymphocyte count was considered to be due to slightly higher white blood cell counts (not statistically significant), whilst absolute lymphocyte counts remained similar to control levels. Other statistically significant changes in haematological parameters were also considered to be of no toxicological relevance as they remained within the range considered normal for rats of this age and strain and/or occurred in the absence of a dose-related trend. These changes included lower white blood cell count of males at 5 mg/kg, lower mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) level of males at 25 mg/kg, and lower partial thromboplastin time (APTT) of females at 25 mg/kg.

CLINICAL BIOCHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and/or
remained within the range considered normal for rats of this age and strain. These changes consisted of a lower albumin and higher total bilirubin and bile acid levels in males at 25 mg/kg, and lower calcium level in males at 5 and 25 mg/kg. The high individual glucose value of one female at 25 mg/kg occurred in the absence of a group response.


MACROSCOPIC EXAMINATION
Necropsy did not reveal any toxicologically relevant alterations. The female at 25 mg/kg that was sacrificed in extremis showed watery-clear fluid in the thoracic cavity, which probably related to a gavage incident (see Mortality). The incidence of other findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend.
These necropsy findings were therefore considered to be of no toxicological relevance, and consisted of enlarged mandibular lymph nodes, a diaphragmatic hernia of the right median lobe of the liver, red discolouration of the mandibular lymph nodes, a red nodule on the epididymal adipose tissue, pelvic dilation of the kidneys, an irregular surface of the forestomach, thickening of the skin of the hindleg, alopecia and fluid in the uterus.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. The statistically significant higher seminal vesicle weight (absolute and relative) of males at 1 mg/kg, and higher liver weight (only statistically significant for relative weight) of females at 25 mg/kg were slight in nature and occurred in the absence of a dose-related trend. These changes were therefore considered to be of no toxicological relevance.

MICROSCOPIC EXAMINATION
The following microscopic findings were considered to be related to treatment with the test substance:
- Duodenum: foamy macrophage foci in the lamina propria were present in 5/5 25 mg/kg (2 minimal, 1 slight, 2 moderate) treated male and 2/6 25 mg/kg (1 minimal, 1 slight) treated female rats.
- Jejunum: foamy macrophage foci in the lamina propria were present in 3/5 5 mg/kg (1 minimal, 2 slight) and in 5/5 25 mg/kg (1 slight, 4 moderate) treated male rats and in 1/5 5 mg/kg (minimal) and in 5/6 25 mg/kg (2 slight, 3 moderate) treated female rats.
- Ileum: foamy macrophage foci in the lamina propria were present in 4/5 5 mg/kg (4 slight) and in 5/5 25 mg/kg (1 minimal, 4 moderate) treated male rats and in 5/5 5 mg/kg (2 minimal, 3 slight) and in 5/6 25 mg/kg (1 minimal, 1 slight, 3 moderate) treated female rats.
- Mesenteric lymph node:
− foamy macrophage foci were present in 2/5 1 mg/kg (1 minimal, 1 slight), in 5/5 5 mg/kg (1 minimal, 3 slight, 1 moderate) and in 5/5 25 mg/kg (3 moderate, 2 marked) treated male rats and in 2/5 1 mg/kg (1 minimal, 1 slight), in 5/5 5 mg/kg (1 minimal, 3 slight, 1 moderate) and in
6/6 25 mg/kg (2 slight, 1 moderate, 3 marked) treated female rats.
− granuloma was present in 1/5 5 mg/kg (minimal) treated male rat, and in 1/5 5 mg/kg (marked) and 1/6 25 mg/kg (slight) treated female rats.
− macrophage foci were present at slightly higher severity in 5 mg/kg treated female rats (3 slight) compared to 3/5 control (3 minimal) and 1/5 1 mg/kg (minimal) treated rats.
The macrophage foci may develop into more vacuolated (foamy) macrophages when taking up more material and finally being organized at higher doses into granulomas. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, and precoital time were unaffected by treatment. The number of corpora lutea and implantation sites showed an apparent trend towards reduced numbers over the dose groups. This was considered to be due to low individual values for corpora lutea and implantation sites for a few animals only, in combination with non-pregnant females (one at 1 mg/kg and two at 25 mg/kg). This apparent trend was therefore considered to be of no toxicological relevance.

GESTATION
Gestation index and duration of gestation were similar between treated and control rats.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: foamy macrophage foci in lamina propria of jejunum, ileum and mesenteric lymph node, granuloma in mesenteric lymph node, and increased absolute and relative neutrophil count at 5 mg/kg bw/d

Results: F1 generation

General toxicity (F1)

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings:

not examined

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

MORTALITY PUPS
Three pups of the control group, two pups at 1 mg/kg, and one pup at 5 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS PUPS
The nature and incidence of blue discolouration of the abdomen shown by one pup of one female of Group 4 remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

BODY WEIGHT PUPS
Body weights of pups were considered to have been unaffected by treatment.

MACROSCOPY PUPS
Two pups found dead showed absence of milk in the stomach. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance..

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at highest dose level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 1 mg/kg/day
Reproduction NOAEL: at least 25 mg/kg/day

Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Tris(hydroxyethyl) oleyl diaminopropane in rats by oral gavage.

The study was based on the following guidelines:

1) OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996.

2) OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

3) OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

4) OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

5) EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

6) OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

7) OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

 

Dose levels are based on the results of a Range Finding and MTD study (NOTOX Project 497209), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 1, 5 and 25 mg/kg.

 

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 1, 5 and 25 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-52 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)), body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

 

Results/discussion

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results:

Treatment resulted in histopathological findings at 25 mg/kg, consisting of foamy macrophage foci in the lamina propria of the duodenum (all males up to moderate degree and 2/6 females up to slight degree), jejunum (all males and 5/6 females up to moderate degree) and ileum (4/5 males at slight degree and in all females up to moderate degree), foamy macrophage foci in the mesenteric lymph node in all males and females (up to marked degree), and granuloma in the mesenteric lymph node in one female (slight).

At 5 mg/kg, these histopathological findings consisted of foamy macrophage foci in the lamina propria of the jejunum (3/5 males up to slight degree and one female at minimal degree) and ileum (4/5 males and all females up to slight degree), foamy macrophage foci in the mesenteric lymph node in all males and females (up to moderate degree), granuloma in the mesenteric lymph node of one male (minimal degree) and one female (marked degree), and a slightly higher severity of macrophage foci in the mesenteric lymph node of females.

Females at 5 and 25 mg/kg also showed higher relative and absolute neutrophil counts, and a trend towards higher white blood cell counts.

At 1 mg/kg, histopathological findings were confined to foamy macrophage foci in the mesenteric lymph node of 2/5 males and 2/5 females (up to slight degree).

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical pathology investigations, macroscopic examination and organ weights).

 

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (25 mg/kg).

No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

 

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (25 mg/kg).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

 

Conclusion

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 1 mg/kg/day

Reproduction NOAEL: at least 25 mg/kg/day

Developmental NOAEL: at least 25 mg/kg/day