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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There are no indications for reproduction toxicity indicated by results form an available reproduction screening study. Possible exposures are very limited due to the characteristics of the substance.

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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

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

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

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

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

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

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.

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

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings:

not examined

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..

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

Reproductive effects observed:

not specified

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and has Klimisch score 1. Information is supported by cross-reading from non-ethoxylated alkyl-diamines.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Effect on fertility via oral route:

Available data from OECD 422 study show no impact to reproduction parameters at all up to highest tested dose of 25 mg/kg bw/day.

 

Oleyl-diamine3EO has been evaluated in a reproduction/developmental toxicity screening test according to OECD 422 guidelines and in compliance to GLP. Four groups of ten male and ten female rats were exposed by oral gavage to Oleyl-diamine3EO at 0, 1, 5 and 25 mg/kg/day. Males were exposed for 29 days, 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.

Reproduction/developmental parameters were evaluated, 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).

No reproduction or developmental 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), or in any of the developmental parameters (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

The study therefore concluded to a reproduction NOAEL of at least 25 mg/kg/day.

In contrast, the parental NOAEL for repeated dose toxicity was found to be 1 mg/kg/day

 

Also cross-reading from non-ethoxylated diamines indicate no concerns for reproduction toxicity as noadverse effects on reproductive organs were identified in the 90 day study in rats on C12-14-diamine. A developmental toxicity study performed on Oleyl-diamine also included endpoints relevant to assessing an effect on fertility. No effects on pre/post implantation rate, late/early resorptions, corpora lutea or number of live fetuses were seen in this study.

Although the available data on Oleyl-diamine3EO does not include information on sperm parameters and oestrus cycle, there are currently no indications of an effect of alkyl-diamines or alkyl-diamine3EO on fertility. In addition, there is no consumer exposure to Oleyl-diamine3EO, and manufacture and industrial use are highly controlled, limiting the possibility of exposures.

Effect on fertility via inhalation route:

Oleyl-diamine3EO is a viscous fluid with a bp > 300°C and has a vapour pressure < 0.0015 Pa at 20°C. Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Effect on fertility via dermal route:

Manufacture and use are highly controlled. Its use is limited to industrial and professional users where its severe corrosive properties will ensure sufficient protection measures to prevent dermal exposure. Furthermore, the substance is not expected to easily pass the skin, and in view of their severe corrosive properties, testing via dermal route for developmental toxicity is not a first choice.

Effects on developmental toxicity

Description of key information

Available data from OECD 422 study show no impact to reproduction parameters at all up to highest tested dose of 25 mg/kg bw/day. Parental NOAEL =1 mg/kg bw

Prenatal developmental toxicity (OECD 414, GLP) based on read-across from Oleyl-diamine: Developmental NOAEL: 20 mg/kg bw, Maternal toxicity NOAEL = 5 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2008-05-14 - 2010-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier S.A.S, Route des Chênes secs-B.P.4105-53941 LE GENEST-ST-ISLE-France
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: within ± 20% of the mean weight (no more information given)
- Housing: The animals were housed individually in IVC cages (except during mating
period where 2 females were paired with one male), type III H, polysulphone cages on Altromin saw fiber bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved or suspended corn oil. The vehicle was chosen as suggested by sponsor and the test item’s solubility. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Lot/batch no. (if required): 058K0070 and 128K0040 (Sigma)

- Administered dose volume: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at specified intervals. Analysis of the dose formulations of the test item in the vehicle (nominal concentration) was performed in the first and last week of the study for all doses. Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. The determination was performed in the first and last week of the study. The dose formulation analysis was performed at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study Nr. 081567.

Details on mating procedure:

- Impregnation procedure: cohoused
- if cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

The test item was administered to sperm positive females (presumed pregnant) from respective GD 0 to GD 19.

Frequency of treatment:

daily

Duration of test:

Duration of test: 20 days (animals were killed on day 20)

Remarks:

Doses / Concentrations:
1.25, 5.0, 20.0 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily during entire gestation period except during weekends and holidays where clinical observation was made only once. Mortality, morbidity, pertinent behavioural changes and all signs of overt toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The sperm positive females were weighed during GD 0, 3, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study.

FOOD CONSUMPTION: Yes
- Food consumption of sperm positive females was measured on respective GD 3, 5, 8, 11, 14, 17 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, ovaries

OTHER:
- Inflammatory Markers:
Serum samples were collected at terminal sacrifice from all females and stored at ≤ -20 °C for the possible analysis of inflammatory markers by ELISA technique.

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Statistical significance for fetal anamolies were determined by Chi Square analysis. Statistical analysis was performed with GraphPad Prism (Version V) software (p < 0.05 was considered as statistical significant).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Test item related clinical signs were observed in high dose females during the entire treatment period. 4 decedents were found (1 in the MD and 3 in the HD group). But the death of only 2 animals might be considered due to toxicity. Statistically significant decrease for the body weight and for the food consumption in the HD group was observed. Also statistical anlysis of parental data revealed significance in the parental parameters gravid uterus weight and adjusted maternal weight in HD group compared to corresponding controls. Decrease in pregnancy rate was observed in HD group (69.56% compared to LD (96%), MD (96.8%) and, control (95.8%)).
The terminally sacrificed animals belonging to the HD group revealed incidences of few lesions at necropsy, which were as gas filled stomach and intestine, whitish spots on adrenals, discoloured liver, small spleen and thymus, enlarged adrenals, bloody lung, discoloured heart, bloody lung.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Few gross abnormalities were seen in fetuses among the control and the treatment groups. Typical external finding noted were protruding tongue,
malrotated limbs, micrognathia, edematous neck, small neck and hematoma (localised). But no statistical deviation was observed for these above findings except for hematoma which were localised and did not show dose related pattern.
Internal observation of the viscera by free hand micro discussion technique revealed range of visceral abnormalities in all groups including control.
However, the statistical analysis revealed differences for findings viz., hemorrhagic kidney-bilateral (HD group), convoluted ureter-bilateral (HD
group), dilated renal pelvis-left side (MD group), split thymus (MD), small spleen (MD group). Most of the above findings (except for hemorrhagic kidney and convoluted ureter in HD group) were not attributed to toxicity due to lack of dose dependent effect.
Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence in both treatment and control groups. The statistical difference observed for supernumerary 14th rib-right side –bilaterally (MD group) and right (HD group), large naso-frontal suture (MD group), incomplete ossification of 4th sternebrum (LD group), split interparietal (HD group) and small hyoid (HD group) were attributed to toxicity, but the statistical difference observed for other anamalies were not considered of toxicological relevance due to lack of dose related pattern.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Summary of clinical observations

Clinical Finding	Group
	C (0 mg/kg)	LD (1.25 mg/kg)	MD (5 mg/kg)	HD (20 mg/kg)
Total number of animals examined	24	25	25	26
Regurgitation	0	1	1	0
Dyspnoea	0	1	0	1
Moving the bedding	0	0	2	1
Salivation	0	0	3	15
Vocalization	0	0	4	9
Sneezing	0	0	0	4
Bloody nasal discharge	0	0	0	1
Weight loss	0	0	0	5
Piloerection	0	0	0	3
Half eye lid closure	0	0	0	1
Apnoea	0	0	0	2
Swollen abdomen	0	0	0	1
Swollen snout	0	0	0	1
Cyanosis	0	0	0	1
Diarrhea	0	0	0	1

Summary of prenatal data

Parameters		C (0 mg/kg)	LD (1.25 mg/kg)	MD (5 mg/kg)	HD (20 mg/kg)
Terminal Body weight (g)	Mean ± SD N	420.17 24.94 23	412.33 38.72 24	408.83 24.85 23	369.13* 53.44 16
Uterus weight (g)	Mean ± SD N	73.16 13.83 23	68.79 26.44 24	74.52 16.57 23	67.5* 24.25 16
Adjusted maternal weight (g)	Mean ± SD N	347.04 20.66 23	343.54 19.42 24	334.3 18.97 23	301.63* 34.13 16
Corpora lutea	Mean ± SD N	14.26 1.48 23	14.88 2.76 24	15.0 2.58 23	15.19 1.42 16
Implantation	Mean ± SD N	12.74 2.14 23	12.63 4.22 24	12.74 3.15 23	13.5 3.2 16
Live Fetuses	Mean ± SD N	12.22 2.26 23	11.75 4.70 24	12.74 3.15 23	11.88 4.5 16
Early resorptions	Mean ± SD N	0.48 0.79 23	0.88 1.03 24	0.52 0.9 23	0.75 1.44 16
Late resorptions	Mean ± SD N	0.04 0.21 23	0.0 0.0 24	0.0 0.0 23	0.88 3.5 16
Total resorptions	Mean ± SD N	0.52 0.79 23	0.88 1.03 24	0.52 0.9 23	1.63 3.59 16
Dead Fetuses	Mean ± SD N	0.0 0.0 23	0.04 0.2 24	0.09 0.29 23	0.0 0.0 16
Sex Ratio (M/F)	Mean ± SD N	2.27 2.37 23	0.96* 0.61 23	1.02* 0.66 22	12.67 3.31 15
Pre-implantation loss	Mean ± SD N	10.86 10.55 23	16.96 22.51 24	15.17 15.3 23	11.07 19.09 16
Post-implantation loss	Mean ± SD N	4.24 6.04 23	11.88 22.48 24	0.0 0.0 23	11.38 25.65 16

Summary of Fetal Visceral Examination

Observations	Group	Control		Low Dose		Mid Dose		High Dose
	Dose	0 mg/kg bw		1.25 mg/kg bw		5 mg/kg bw		20 mg/kg bw
	No. Of litters evaluated	23		24		23		16
	No. Of pups evaluated	147		147		152		99
		A	B	A	B	A	B	A	B
Hemorrhagic Kidney (B)		0	0.0	1*	0.75	3	2.11	5	5.49
Convoluted Ureter (B)		7	5.19	11	8.21	16	11.27	15*	16.48
Dilated Renal Pelvis (L)		0	0.0	3	2.24	5*	3.52	0	0.0
Thymus		0	0.0	2	1.49	4*	2.82	1	1.1
Small Spleen		2	1.48	2	1.49	9*	6.34	2	2.2

Summary of Fetal Skeletal Examination

Observations	Group	Control		Low Dose		Mid Dose		High Dose
	Dose	0 mg/kg bw		1.25 mg/kg bw		5 mg/kg bw		20 mg/kg bw
	No. Of litters evaluated	23		24		23		16
	No. Of pups evaluated	147		147		152		99
		A	B	A	B	A	B	A	B
Supernumerary rib-14th T (B)		6	4.08	10	6.8	24*	15.79	4	4.04
Supernumerary rib-14th T (R)		2	1.36	8	5.44	8	5.26	6*	6.06
Large-NasoFrontal Suture		0	0.0	1	0.68	5*	3.29	0	0.0
IO-4th Sternerbum		0	0.0	5*	3.4	1	0.66	0	0.0
Split-Interparietal		15	10.2	13	8.84	18	11.84	19*	19.19
Small-Hyoid		0	0.0	2	1.36	3	1.97	3*	3.03

Conclusions:

Based on the findings, the NOAEL (No observed adverse effect level) for maternal toxicity is believed to be 1.25 mg/ kg body weight based on clinical observations seen in some animals at 5 mg/kg, whereas the NOAEL for embryo-fetal toxicity is believed to be 20 mg/ kg body weight.

Executive summary:

This Prenatal developmental toxicity study of N-Oleyl-1,3-diaminopropane was conducted in pregnant female Wistar rats to detect the possible adverse effect on pregnant females and embryofetal development when administered by oral gavage from respective gestation day 0 to 19.

Nulliparous and non pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on day of positive vaginal smears (GD 0). Four groups of presumed pregnant females were dosed daily by oral gavage with 1.25, 5 and 20 mg/kg body weight per day of N-Oleyl-1,3-diaminopropane at dose volume of 4 mL/kg body weight. Control animals were handled identically as treated groups and received vehicle in similar volume as treated groups. The test item formulation was prepared freshly and dose volumes were adjusted based on the most recent body weight measurement. Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured on various gestation days. The treated and control females were sacrificed on respective gestation day 20.

Followed by the gross necropsy evaluation of the females, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead fetuses. Fetuses were identified by colour strings, sexed and weighed. All fetuses were observed for the external abnormalities, half of the fetuses for the visceral abnormalities, craniofacial examination and remain half of the litter for skeletal abnormalities. Uteri of the non pregnant females were processed with 0.5 % ammonium sulphide solution and checked for the early embryonic deaths if any.

 

Results:

Test item related clinical signs were observed in high dose females during the entire treatment period. Also effects on clinical observations were observed in animals of the MD group.

However, there were four decedents in this study [1 in MD group (Animal 62) and 3 in HD group (Animals 80, 82 and 98)]. Animal no. 82 was euthanised for humane reason. The death of two animals (Animals 62 and 98) were considered due to gavaging error and other two (Animals 80 and 82) might be considered due to toxicity. 

Statistically significant decrease was observed for body weight and body weight change throughout the gestation period in HD group.  Statistically significant decrease in overall food consumption was observed in HD group compared to corresponding control. Statistical analysis of prenatal data revealed significance in prenatal parameters like gravid uterus weight and adjusted maternal weight in HD group compared to corresponding controls. No other prenatal parameters like No. of corpora lutea, implantations percent preimplantation loss, group mean number of live fetuses, early resorptions, late resorptions, total resorptions, group mean number of female fetuses, sex ratio (M/F) and percent post implantation loss showed statistical deviation compared to corresponding controls.

 

Statistically significant difference was observed for group male litter weight (LD group), sex ratio (LD and MD groups) and total number of male fetuses (LD group) compared with controls. These findings were not attributed to toxicity as no dose related pattern was observed. Decrease in pregnancy rate was observed in HD group (69.56%) as compared to LD (96%), MD (95.8%), and control (95.8%).

Few gross external abnormalities were seen in fetuses among the control and treatment groups. Typical external findings noted were protruding tongue, malrotated limbs, micrognathia, edematous neck, small neck and hematoma (localised).But no statistical deviation was observed for these above findings except for hematoma which were localised and did not show dose related pattern.

Internal observation of the viscera by free hand micro discussion technique revealed range of visceral abnormalities in all groups including control. However, the statistical analysis revealed differences for findings viz., hemorrhagic kidney-bilateral (HD group), convoluted ureter-bilateral (HD group), dilated renal pelvis-left side (MD group), split thymus (MD), small spleen (MD group). Most of the above findings (except for hemorrhagic kidney and convoluted ureter in HD group) were not attributed to toxicity due to lack of dose dependent effect.

Craniofacial examination by razor blade serial sectioning technique revealed no statistical significant difference for any of the findings observed in treatment and control groups. 

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence in both treatment and control groups. The statistical difference observed for supernumerary 14th rib-right side-bilaterally (MD group) and right (HD group), large naso-frontal suture (MD group), incomplete ossification of 4th sternebrum (LD group), split interparietal (HD group) and small hyoid (HD group) were attributed to toxicity, but the statistical difference observed for other anomalies were not considered of toxicological relevance due to lack of dose related pattern.

The terminally sacrificed animals belonging to the HD group revealed incidences of few lesions at necropsy, which were as gas filled stomach and intestine, whitish spots on adrenals, discoloured liver, small spleen and thymus, enlarged adrenals, bloody lung, discoloured heart, bloody lung. The finding like dark coloured food rest in caecum observed in most of the animals of control and treatment groups cannot be considered as toxicity related and in most of the animals this finding observed was not reported.

 

Based on the findings, the NOAEL (No observed adverse effect level) for maternal toxicity is believed to be 1.25 mg/ kg body weight based on clinical observations seen in some animals at 5 mg/kg, whereas the NOAEL for embryo-fetal toxicity is believed to be 20 mg/ kg body weight.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and has Klimisch score 1. Although based on Oleyl-diamine, the results are relevant for Oleyl-diamine3EO.

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

Oleyl-diamine3EO has been evaluated in a reproduction/developmental toxicity screening test according to OECD 422 guidelines and in compliance to GLP.

Four groups of ten male and ten female rats were exposed by oral gavage to Oleyl-diamine3EO at 0, 1, 5 and 25 mg/kg/day. Males were exposed for 29 days, 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.

Reproduction/developmental parameters were evaluated, 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).

No reproduction or developmental 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), or in any of the developmental parameters (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

The study therefore concluded to a reproduction NOAEL of at least 25 mg/kg/day.

In contrast, the parental NOAEL for repeated dose toxicity was found to be 1 mg/kg/day

 

Additional information on developmental toxicity of Oleyl-diamine3EO can be derived by cross-reading from the non-ethoxylated diamine Oleyl-diamine. For this substance a full developmental toxicity study is available according to OECD 414 guidelines in compliance to GLP.

Cross-reading from this substance is acceptable on the basis of similarities of structure and properties leading to common biological activity.

This study was performed by dosing pregnant rats from day 0-19 of gestation with either 0, 1.25, 5 or 20 mg/kg bw of Oleyl-diamine. The animals were then sacrificed and the fetuses subject to external, visceral and skeletal examination. There was a slight increase in visceral and skeletal variations. No malformations were observed. Neither of the variations are considered to be of toxicological relevance as there were no dose-response pattern. Maternal toxicity including deaths in the high dose group, reduced bodyweight gain and reduced pregnancy rate were observed.

The NOAEL (No observed adverse effect level) for maternal toxicity is believed to be 1.25 mg/ kg body weight based on clinical observations seen in some animals at 5 mg/kg, whereas the NOAEL for embryo-fetal toxicity is believed to be 20 mg/ kg body weight.

 

The conclusion that alkyl-diamine3EO is not teratogenic is supported by information that was submitted for TSCA 8(e)involving an illegible copy of an old study report. Adequate assessment of the data is not possible leading to a low validity.

However, this study concluded that treatment of pregnant NZW rabbits during gd 6-18 with ethoxylated tallow diamine at dose levels of 0, 1, 5 and 25 mg ai/kg bw/d did not cause any increase in major malformations, minor anomalies or skeletal variants. Based on the findings above, the test material is embryotoxic at maternally toxic doses, but is not teratogenic.

Effect on developmental toxicity: via inhalation route:

Oleyl-diamine3EO is a viscous fluid with a bp > 300°C and has a vapour pressure < 0.0015 Pa at 20°C. Its use is limited to industrial and professional users and does not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Effect on developmental toxicity: via dermal route:

Manufacture and use are highly controlled. Its use is limited to industrial and professional users where its severe corrosive properties will ensure sufficient protection measures to prevent dermal exposure. Furthermore, the substance is not expected to easily pass the skin, and in view of their severe corrosive properties, testing via dermal route for developmental toxicity is not a first choice

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, reproduction toxicity is not expected. The observed effects are local, reflecting a point-of-first-contact effect.

The mode of action of for follows from its structure, consisting of an apolar fatty acid chain and a polar part of an ethoxylated amine.In physiological circumstances the nitrogens can become positively charged, resulting to a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. At pH below 7.2 more than 99.9% of the substance has at least one positively charged nitrogen. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures.Cytotoxicity through disruption of cell membrane at exposure site will occur rather than absorption over the cell membrane. Consequently, significant uptake followed by placental transfer is not expected to occur.

Justification for classification or non-classification

Available data from a reproduction screening study (OECD 422) with Oleyl-diamine3EO showed no effects on reproduction parameters. Also, cross-reading from a developmental toxicity (OECD 414) with Oleyl-diamine indicate no concerns for developmental toxicity. In both studies the lack of developmental effects at the highest dose levels tested of 25 resp. 20 mg/kg bw/day is in large contrast with the NOAELs obtained for parental toxicity of 1 resp. 5 mg/kg bw.

The available data do not indicate a concern for reproductive health.

 

For fertility firm conclusion regarding classification cannot be made, as a full 2-generation reproduction study is lacking. But based on limited exposures by dermal route (substance is severely irritating/ corrosive) or by inhalation (very low vapour pressure), as well as lack of indication for concerns regarding reproductive toxicity from repeated dose studies and a developmental toxicity study there are no concerns and further testing is not indicated.