Registration Dossier

Administrative data

Description of key information

Ethanol, 2, 2’-iminobis-, N-coco alkyl derives CAS No 61791-31-9 will be registered for REACH as 2, 2’-(C12-18 evennumbered alkyl imino) diethanol CAS No 71786-60-2.

A 14 day dose ranging gavage study was carried tout to anable the dose levels to be selected for a levels for an OECD guideline 422 (Gavage) Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (adopted 22 March 1996).

From this OECD 422 study an NOAEL for repeat dose (42 day) systemic toxicity was derived.

Additional testing was required as 2, 2’-(C12-18 evennumbered alkyl imino) diethanol is a high tonnage Annex X substance, ECHA required an OECD408, 90-day (13 week) oral study in rats. A NOAEL for repeat dose toxicity was also derived from the study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 16 October 2009 and 08 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:HsdRccHan™:WIST strain rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test Animals
- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
297 to 342g (male); 184 to 233g (female)
- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

- Diet:
The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Water:
Water intake was measured and recorded daily for each cage group (with the exception of non-recovery (satellite) animals during the mating phase). Individual daily water intakes were measures for females during the gestation and lactation phases of the study

- Acclimation period:
For 12 days

ENVIRONMENTAL CONDITIONS

- Temperature:
21 ± 2 °C

- Humidity:
55 ± 15 %

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
20 October 2009 and 15 December 2009 (including recovery phase animals)

Route of administration:
oral: gavage
Vehicle:
other: Arachis oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 0142-0416). Results from the previous study showed the formulations to be stable for at least twenty days. Formulations were therefore prepared twice monthly during the treatment period and stored at approximately +4ºC in the dark, under nitrogen.
Samples of each test material formulation were taken and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within plus or minus 9% of the nominal concentration.

DIET PREPARATION
- Not applicable

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Arachis oil BP

- Justification for use and choice of vehicle (if other than water):
Not applicable

- Concentration in vehicle:
31.3, 7.5 and 2.5 mg/ml

- Amount of vehicle (if gavage):
4 ml/kg bodyweight

- Lot/batch no. (if required):
Not applicable

- Purity:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml. Procedural recoveries were performed at each dose level on every analysis occasion.
Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml. The test material formulations were sampled and analysed within five days of preparation. The results indicate that the prepared formulations were within +9% of the nominal concentration.

See attached Appendix 26 - Chemical Analysis of Test Material Formulations, Methods and Results
Duration of treatment / exposure:
The oral administration of the test substance to rats for a period of up to fifty-four consecutive days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
Dose levels of 10, 30 and 125 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg/day – control: 10 animals per sex.
10 mg/kg/day : 10 animals per sex.
30 mg/kg/day : 10 animals per sex.
125 mg/kg/day : 10 animals per sex.
Recovery (Satellite ) 0 mg/kg/day – control: 5 males only.
Recovery (Satellite ) 125 mg/kg/day : 5 males only.
Control animals:
yes, concurrent vehicle
Details on study design:

- Dose selection rationale:
Based on Preliminary Fourteen Day Repeated Dose Oral (Gavage) Range-Finder in the Rat

- Rationale for animal assignment (if not random):
Random

- Rationale for selecting satellite groups:
To determine potential regression of any detected systemic responses elicited by administration of the test material

- Post-exposure recovery period in satellite groups:
Fourteen days

- Section schedule rationale (if not random):
Random
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:

- Yes see attached tables and appendices

- Time schedule:

- Immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, thirty minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during
parturition where applicable). During the treatment-free period, recovery males were observed once daily. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes (see above).
- Time schedule: As above.

NEUROBEHAVIOURAL EXAMINATION:

- Yes see attached tables and appendices

- Functional Observations were performed prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of
functional/behavioural toxicity.

- Functional performance tests (motor activity, forelimb/hindlimb grip strength and sensory reactivity) were also performed on five selected males
during the final week of treatment and five Day 4 post partum females from each dose level.

BODY WEIGHT:

- Yes see attached tables and appendices

- Time schedule for examinations:

- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating w as evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Bodyweights were
also recorded prior to termination

- For parameters checked see attached Tables.

FOOD CONSUMPTION:

- Yes see attached tables and appendices

- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating
phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of adults throughout the study period.

- FOOD EFFICIENCY:

- Yes see attached tables

- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period, and for females prior to mating.

WATER CONSUMPTION:

- Yes see attached tables

- Water intake was measured gravimetrically and recorded daily for each cage group (with the exception of non-recovery animals during the mating - phase). Individual daily water intakes were measured for females during the gestation and lactation phases of the study.

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:

- Yes see attached tables and appendices

- Time schedule for collection of blood:

- Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). These investigations were also performed on all recovery
(satellite) males at the end of the treatment-free period (Day 56).

- Blood samples were obtained from the lateral tail vein or by cardiac puncture at termination, if applicable.

- Anaesthetic used for blood collection:
- No

- Animals fasted:
- No

OTHER:

MATING

- Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each
morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal
smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to
their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and
lactation.

PREGNANCY AND PARTURITION

- Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations
were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

LITTER SIZE

On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1.
For each litter the following was recorded:

i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4
post partum
iii) Clinical condition of offspring from birth to Day 5 post partum
iv) Individual offspring weights on Day 1 and 4 post partum (litter weights were calculated retrospecively from offsring weights).

PHYSICAL DEVELOPMENT

All live offspring were assessed for surface righting reflex on Day 1 post partum.

- see attached tables and appendices


















Sacrifice and pathology:
GROSS PATHOLOGY:
- Yes see attached Table 21 and 22 and Appendices 246 to 249 for males and 250 to 253 for females

HISTOPATHOLOGY:
Yes see attatched Table 23 and Appendix 24
Other examinations:
MORTALITY DATA
- Yes (no unscheduled deaths ocured during the study)
ORGAN WEIGHTS
- Yes see attached Tables and Appendices
Statistics:
The volume of statistical references exceeds the storage capacity in this section it has therefore been included as an attachment titled 0142-0417 Statistics.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Mortality.

No unscheduled deaths were detected.

Clinical Observations.

A higher incidence of increased salivation was detected soon after dosing and up to one hour after dosing for animals of either sex treated with 125 and 30 mg/kg/day, and also for males treated with 10 mg/kg/day when compared to controls. Regression was evident following the cessation of treatment in recovery 125 mg/kg/day males.

Functional Observations.

No treatment-related effects were evident in the weekly behavioural assessments, sensory reactivity, grip strength or motor activity.

Bodyweight.

No adverse effect on bodyweight change was detected for males or for females during the pre-mating and gestation phases. Lower bodyweight gains were evident for females treated with 125 mg/kg/day when compared to controls during the lactation phase of the study. No adverse effects were evident at 30 or 10 mg/kg/day.

Food Consumption.

No adverse effects on dietary intake were evident for males or for females during the pre-mating or gestation phases of the study. A slight reduction in dietary intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation.

Water Consumption.

No overt intergroup differences in water intake were detected for males or for females during the pre-mating or gestation phases of the study. A reduction in water intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation.

Reproductive performance.

Mating.

No treatment-related effects were detected in mating performance.

Fertility.

No treatment-related effects were detected in fertility.

Gestation.

No treatment-related effects were detected on gestation length.

Litter responses.

Litter size and Viability.

Lower litter sizes, live birth indices and reduced numbers of viable litters were evident at 125 mg/kg/day when compared to controls. Slightly lower numbers in corpora lutea and implantation sites were evident for females treated with 125 mg/kg/day when compared to controls, and higher post-implantation losses were also evident.

Offspring Growth and Development.

Lower total litter weights were evident at 125 mg/kg/day in comparison to control values. Bodyweights and surface righting assessments were not
affected.

Laboratory Investigations.

Haematology.

Males treated with 125 mg/kg/day showed a reduction in haemoglobin, haematocrit, mean cell haemoglobin, mean cell volume and reticulocyte counts when compared to controls. These findings were considered to be of no toxicological significance.

No treatment-related effects were evident for females treated with 125 mg/kg/day, or for animals of either sex treated with 30 and 10 mg/kg/day.

Blood Chemistry.

No significant effects were detected in the blood chemical parameters investigated.

Pathology.

Organ Weights.

Males treated with 125 mg/kg/day showed slightly higher absolute and bodyweight-relative spleen and liver weights. These findings were considered to be of no toxicological importance.

No treatment-related effects were detected for females treated at 125 mg/kg/day, or for animals of either sex treated with 30 or 10 mg/kg/day.

Necropsy.

Offspring: No treatment-related macroscopic abnormalities were detected for offspring from treated animals when compared to control litters.

Adults: Treatment-related findings were confined to the presence of a thickened non-glandular region of the stomach for one male treated with
125 mg/kg/day.

Histopathology. The following treatment-related changes were observed:

STOMACH: Acanthosis, frequently with associated hyperkeratosis, was seen in the forestomach of all animals of either sex treated with 125 mg/kg/day, and in males treated with 30 mg/kg/day. There was evidence of regression of the condition in recovery 125 mg/kg/day males following an additional fourteen days without treatment.

Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
30 other: mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity.
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
other: Reproductive toxicity
Sex:
female
Basis for effect level:
other: Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day. A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity
Critical effects observed:
not specified

See attached (0142-0417) Tables, Figures, Appendices, Addenda and Statistics.

 

The following results refer to theFourteen day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Part 2) with Assessment of Maximum Tolerated Dose (Part 1)

 

Part 1:Maximum Tolerated Dose

 

Mortality.

One 500 mg/kg/day female was found dead on Day 3 and the two remaining females were killedin extremison Day 3. There were no further unscheduled mortalities.

 

Clinical Observations.

No clinical observations were detected at 50 mg/kg/day and at 100 mg/kg/day findings were confined to one instance of transient increased salivation. At 200 mg/kg/day, increased salivation was detected in all animals. At 500 mg/kg/day, increased salivation was detected and accompanied on occasions by hunched posture and pilo-erection, tiptoe gait and ano-genital staining, lethargy and ptosis. All animals then showed a decline in general condition leading to the death of one animal and a decision was then taken on humane grounds to sacrifice the two surviving animals. Animals subsequently treated at 350 mg/kg/day displayed findings of increased salivation, diuresis, hunched posture, pilo-erection, decreased respiration, emaciation, ptosis, lethargy and pallor and diarrhoea.

 

Bodyweight.

Bodyweight gains were evident for females treated with 50 mg/kg/day. One female treated with 100 mg/kg/day showed a bodyweight loss of 1g on Day 3 although all females showed bodyweight gains on Day 5. One female treated with 200 mg/kg/day showed an 8g bodyweight loss on Day 3 and another female treated with 200 mg/kg/day showed a bodyweight loss of 5g on Day 5. Bodyweight gains at this dose level were less than those observed at 50 and 100 mg/kg/day. Bodyweight gains were evident for two females treated with 350 mg/kg/day on Day 3 and the bodyweight for one female was unchanged on Day 3 compared to the Day 1 bodyweight. Bodyweight losses of 3g and 29g were evident for two females treated with 350 mg/kg/day on Day 5. At 500 mg/kg/day, all females showed bodyweight losses of between 6 and 16g prior to treatment on Day 3, and further bodyweight losses of 1g and 6g were evident for the remaining two females prior to termination.

 

Necropsy.

The female treated with 500 mg/kg/day found dead on Day 3 showed a distended stomach, and sloughing of the glandular and non-glandular gastric epithelia. Gaseous distension was also observed in the small and large intestines. The remaining two females treated with 500 mg/kg/day and terminated on Day 3 showed gaseous distension of the gastro-intestinal tract. Females treated with 350 mg/kg/day were terminated following five days of treatment. One female (number 5) showed gaseous distension of the gastro-intestinal tract and sloughing of the non-glandular gastric epithelium. The remaining two females treated at this dose level did not reveal any macroscopic abnormalities.

 

Conclusion.

Oral administration of the test material to rats for up to five days at dose levels between 50 and 500 mg/kg/day resulted in significant toxicity at 500 and 350 mg/kg/day. The Maximum Tolerated Dose was therefore considered to be between 200 and 300 mg/kg/day.

 

Part 2: Fourteen day Repeated Dose Oral (Gavage) Range-Finding Toxicity StudyResults:

 

RESULTS

 

Mortality

Animals of either sex treated with 250 mg/kg/day were killedin extremison Day 10 following substantial bodyweight losses and a decline in physical health. There were no further unscheduled deaths.

 

Clinical Observations

One male treated with 250 mg/kg/day displayed increased salivation and noisy respiration soon after dosing from Day 1 and one female treated at this dose level displayed post-dose increased salivation from Day 2. Another female displayed diarrhoea on Days 3 and 4. Diarrhoea was also evident for one male on Day 3 and this male was observed as hunched on Day 4 and from Day 7 onwards. Incidents of increased salivation were also evident for remaining males from this dose group, from Day 2 and staining around the ano-genital region, suggestive of diarrhoea was observed in a number of animals of either sex from Day 4. On the morning of Day 10, clinical signs of lethargy, hunched posture, dehydration and diarrhoea were observed for all animals.

 

These clinical signs, together with bodyweight losses was considered excessive and the animals treated at this dose level were terminated on Day 10. Increased salivation was detected soon after dosing for animals of either sex treated with 150 mg/kg/day between Days 5 to 14. One male also displayed noisy respiration, although this was confined to Day 12 only.

 

No macroscopic abnormalities were detected for animals of either sex treated with

75 mg/kg/day.

 

Bodyweight

Substantial losses in bodyweight were evident for animals of either sex treated with 250 mg/kg/day, with the effect more prominent in males, which resulted in statistically significantly differences in this dose group when compared to control values (P<0.01).

 

These substantial bodyweight losses and the clinical signs observed, resulted in the

termination of this dose group on Day 10. Slight bodyweight losses were also evident for females treated with 150 mg/kg/day between Days 1 and 4 resulting in statistically significant reductions when compared to controls (P<0.05), although improvement was evident thereafter. The overall gain during the treatment period at 150 mg/kg/day was only slightly lower than controls (males -3.8%, females -2.9%), therefore, this was not considered to represent an adverse effect of treatment.

 

No adverse effect on bodyweight change was evident for animals of either sex treated with 75 mg/kg/day. Females treated with 75 mg/kg/day showed a statistically significant reduction in bodyweight gains (P<0.05), although this was only observed during the final four days of treatment.

 

Food Consumption

A reduction in dietary intake was evident for animals of either sex treated with 250 mg/kg/day prior to their early sacrifice on Day 10.

Slight reductions in dietary intake (approximately 11%) were also evident for animals of either sex treated with 150 mg/kg/day when compared to control values over the fourteen day treatment period. These reductions were considered not to represent an adverse effect of treatment.

 

No adverse effects on dietary intake were evident for animals of either sex treated with 75 mg/kg/day.

 

Water Consumption

Increases in water consumption were evident for animals of either sex treated with 250 mg/kg/day when compared to controls during the nine days of dosing, prior to their early sacrifice.

 

No adverse effects on water intake were evident for animals of either sex treated with 150 or 75 mg/kg/day.

 

Organ Weights

Animals of either sex treated with 150 mg/kg/day showed a statistically significant increase in absolute and relative liver weights when compared to controls (P<0.01).

 

The effect extended into the 75 mg/kg/day dose group, although statistical significance was only achieved for males (P<0.01).

 

Necropsy

 

The following macroscopic findings were observed for the high dose group terminated on Day 10: one male displayed pale lungs, a thickened urinary bladder with pale coloured contents, raised limiting ridge of the stomach, and a thickened and sloughing nonglandular region of the stomach. Another male showed reddened lungs and stomach changes consisting of gaseous distension, sloughing of the non-glandular region and a reddened appearance. The third male displayed a thickened non-glandular region which also showed sloughing. Sloughing of the non-glandular region was also evident for two females, one of which also showed a raised limiting ridge. One interim death female did not show any macroscopic abnormalities.

Two males and one female treated with 150 mg/kg/day displayed a thickened nonglandular region of the stomach, which was also evident for one female treated with 75 mg/kg/day.

 

No macroscopic abnormalities were detected for males treated with 75 mg/kg/day.

 

DISCUSSION

 

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage for a period of up to fourteen consecutive days at dose levels of 250, 150 and 75 mg/kg/day resulted in treatment-related effects at all dose levels.

 

Clinical signs were observed at the highest dose level. These included increased salivation, noisy respiration, staining around the ano-genital region, diarrhoea and hunched posture. By Day 10, all animals showed lethargy, hunched posture, dehydration and diarrhoea. Significant bodyweight losses were also evident in this dose group, together with an increase in water intake and reduced dietary intake. Due to the severity of these effects, this dose level was considered excessive and the dose group was terminated on Day 10.Post-mortemexaminations revealed a number of effects, the most noticeable were stomach changes including raised limiting ridge, and thickened and sloughing of the gastric epithelia.

 

Clinical signs at 150 mg/kg/day were confined to isolated instances of increased salivation soon after dosing and noisy respiration. Slight bodyweight losses were evident for females during the first four days of treatment, although overall bodyweight change in this dose group was not adversely different from control values. There were no adverse effects on dietary intake detected at this dose level, althoughpost-mortemfindings revealed an increase in absolute and bodyweight-relative liver weights when compared to controls. Macroscopic examinations also revealed thickened non-glandular gastric epithelia for three animals from this treatment group.

 

Treatment-related effects at 75 mg/kg/day were confined to increases in absolute and bodyweight-relative liver weights, which were observed for animals of either sex, and a thickened non-glandular region of the stomach, which was observed for one female during thepost-mortemexaminations.

 

CONCLUSION

 

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31- 9) to rats by gavage for a period of up to fourteen consecutive days at dose levels of 250, 150 and 75 mg/kg/day resulted in significant toxicity at 250 mg/kg/day.

 

Treatment-related effects including increased liver weights and macroscopic gastric changes were also evident at 150 and 75 mg/kg/day; therefore a ‘No Observed Effect Level’ (NOEL) was not established at the dose levels employed in the fourteen day range-finding phase.

 

 

 

Conclusions:
The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment-related effects at 125 and 30 mg/kg/day. These effects consisted of a localised irritant effect, and almost complete regression was evident following the treatment-free period. This change may be considered to be an adverse event and of importance when considering the impact on reproductive performance. Based upon the histopathological changes observed in the stomach, a “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity, was considered to be 30 mg/kg/day.
Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day. A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity.

In the study the controls showed a mean of 19.4 corpora lutea, which is outside the historical control range of 10-18. While the 125 mg/kg/day top dose females did show a reduced number of corpora lutea at a mean of 15.2 this was still well within the historical control range as was the number of implantations sites and there were no indications of a dose response. Due to the inevitable variability in the counting of corpora lutea in females four days after littering and the lack of a dose response these differences are not considered to be evidence of a toxic effect on reproduction.

The effects on the reproductive parameters were only seen in the 125 mg/kg bodyweight /day group, with no indication of a dose response at the lower doses. There was a combination of effects seen in the parental females in particular in the two litters which showed some of the most marked effects on the offspring survival which could be explained as possibly being secondary effects of maternal toxicity. The increase in post implantation loss was a more widespread phenomenon, however the marked increase was again influenced by females 71 and 72 which showed 9 and 5 post implantation losses and in addition female 77 which showed 11 post-implantation losses and produced no offspring. The remaining females in the 125 mg/kg bodyweight /day group while overall they still showed some increased post implantation loss individually the highest loss was 4 out of 14 which compared to one negative control female that showed 4 losses out of 17 implantation sites. As these effects on post implantation loss are concentrated particularly in three females it is possible that this is related to a toxic effect in those parental females rather than a more specific dose related development toxic effect in this group.

Due to the limitations of the study design of the OECD422 it is considered as a screening test for reproductive toxicity. It is not able to elucidate definitive reproductive effects particularly where they may involve developmental toxicity. Based on the findings in this study which indicate the possibility of foetal toxicity a full developmental toxicity study OECD 414 will be required to establish if these finding represent genuine developmental toxicity (foetal toxicity).
Executive summary:

ORAL (GAVAGE) COMBINED REPEAT DOSE TOXICITY STUDY WITH REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST IN THE RAT (OECD 422 1996). PROJECT No. 0142-0417

 

Introduction.

The study was performed according to the protocol presented in Appendix 25 and was designed to screen for potential adverse effects of the test material on reproduction, including offspring development, following repeated oral administration to the Wistar Han™:HsdRccHan™:WIST strain rat for up to forty-five days(including a two week maturation phase, pairing, gestation and early lactation),at dose levels of 10, 30 and 125 mg/kg/day. A control group was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males, were treated with the high dose (125 mg/kg/day) or the vehicle alone for forty-two days and then maintained without treatment for a further fourteen days.

 

The study was designed to comply with the OECD Guidelines for Testing of Chemicals No. 422“Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

Methods.

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to forty-five consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 10, 30 and 125 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males, were treated with the high dose (125 mg/kg/day) or the vehicle alone for forty-two days and then maintained without treatment for a further fourteen days.

 

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group. 

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

 

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

 

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Conclusion.

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment-related effects at 125 and 30 mg/kg/day. These effects consisted of a localised irritant effect, and almost complete regression was evident following the treatment-free period. This change may be considered to be an adverse event and of importance when considering the impact on reproductive performance. Based upon the histopathological changes observed in the stomach, a “No Observed Adverse Effect Level” (NOAEL) for systemic toxicity, was considered to be 30 mg/kg/day.

 

Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day.  A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity.

 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 06 December 2017 Experimental completion date: 06 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Test item Ethanol, 2,2’-iminobis-, N-C12-18-alkyl derives

2,2’-(C12-18 even-numbered alkyl imino)diethanol.
Bis(2-hydroxyethyl)cocoalkylamine
CAS number 71786-60-2
Intended use Substance used in industry.
Appearance Light yellow liquid.
Storage conditions At ambient temperature (15 to 25C), in the dark.
Supplier Sponsor.
Batch number 1373142
Expiry date 28 November 2018
Purity 98%
Supplier’s responsibilities Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST ) strain was used because of the historical control data available at this laboratory
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals
Strain/Species
RccHan™;WIST rat.

Supplier
Envigo RMS Limited.

Number of animals
44 males and 44 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 12 days before commencement of treatment.

Age of animals at start of treatment 41 to 47 days.

Weight range of the animals at the start of treatment Males: 124 g to 175 g
Females: 115 g to 145 g

Allocation and Identification
Allocation Randomly allocated on arrival.

Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Replacement before treatment commenced
Ocular abnormalities One female

On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed 20% of the mean for the appropriate sex. No replacements were necessary.

Animal Care and Husbandry
Environmental Control
Animal facility
Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply
Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity
Monitored and maintained within the range of 20-24ºC and 40-70%.

There was one minor deviation from the range (19ºC on Day 67). This deviation was minor and of short duration and was not considered to have influenced the health of the animals and/or the outcome of the study.

Lighting
Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply
Public supply with automatic stand-by generators.

Animal Accommodation
Cages
Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.

Cage distribution
Males and females were blocked by group and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.

Number of animals per cage
Three or four of the same sex.

Bedding
Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen chew block Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C Diet.
Availability Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during the period of urine collection).
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by oral gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
arachis oil
Details on oral exposure:
Route
Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at
Constant doses in mg/kg/day.

Volume dose
4 mL/kg body weight.

Individual dose volume
Calculated from the most recently recorded scheduled body weight.

Control (Group 1)
Vehicle at the same volume dose as the treated groups.

Frequency
Once daily at approximately the same time each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Liquid formulation
The homogeneity and stability of formulations during refrigerated storage (2 to 8°C) was confirmed and supplied by the Sponsor (Harlan Project Nos. 0142-0416 and 0142 0417) however, no documented stability during ambient temperature was included as part of these trials. Therefore, a trial to assess the stability and homogeneity of the test item in the liquid matrix at ambient temperate (15 to 25°C) for up to four hours was conducted as part of this study.



Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2.5 and 250 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration
Samples of each formulation prepared for administration in Weeks 1 and 13 of treatment were analyzed for achieved concentration of the test item.

Preparation of Calibration Standards
A primary standard solution (500000 ng/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in propan-2-ol (100 mL). A econdary standard solution (10000 ng/mL) was prepared by appropriate dilution of the primary standard using diluent (10 mL). A tertiary standard solution (1000 ng/mL) was prepared by appropriate dilution of the secondary standard using diluent to the final volume of 10 mL.

Solutions for instrument calibration were prepared by appropriate dilution of the tertiary standard using diluent and contained Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives at nominal concentrations of 10 ng/mL, 20 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 80 ng/mL and 100 ng/mL.

Calibration solutions were injected onto the LC-MS/MS, at the beginning of each sample analysis sequence, using the conditions detailed in the chromatographic section. The standard prepared at 50 ng/mL was injected in duplicate to bracket every three samples.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using ultrasonic vibration in a suitable volume of propan-2-ol. The extract was then diluted further with propan-2-ol with final dilutions performed using diluent, to provide a solution containing Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives at an expected concentration within the range 20 ng/mL to 80 ng/mL.

The concentration of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in the final solution was quantified by LCMS/MS as detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (arachis oil) with known amounts of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives. The prepared procedural recoveries were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
High performance liquid chromatograph and mass spectrometer (LC-MS/MS): Agilent 1100 pump, Perkin Elmer PE200 Autosampler and Sciex API 3000 equivalent mass spectrometer

Column: Agilent Poroshell SB C18, 2.7 μm, 100 x 4.6 mm

Column temperature: 45ºC

Sample temperature: Ambient

Mobile Phase A: 0.1M ammonium acetate(aq)/Acetic acid 100/0.1 v/v

Mobile Phase B: MeOH / Acetic Acid 100/0.1 v/v

Flow rate: 0.5 mL/minute

Ionisation: Turboionspray – positive ion mode

Source temperature: +500°C

Collision gas: Nitrogen

Collision energy: 35 eV

Dwell time: 100 ms

Pause time: 5 ms

Ions to be monitored: m/z 274.5 to m/z 106.1 (used for quantitate)

Run Time: 12 minutes

Approximate Retention time: 8.1 minutes

Calculations
The peak counts response for Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area counts of the peak observed at the characteristic retention time for Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives in sample and procedural recovery chromatograms was measured. The concentration of Ethanol, 2,2’-Iminobis-, N-C12-18-Alkyl Derives was determined using the following equation:

Determine the response factor for the single standard.

Response factor (RF) = Calibration standard peak response (Ac ) / Concentration of calibration standard (ng/mL)

Then for each sample chromatogram determine the concentration of the test item using the following equations:

Analysed concentration (mg/mL) = (As/RF) x (V/W) x (D/1,000,000)

Procedural recovery values were determined using the following equation:

Procedural recovery = (Analyzed concentration/ Fortified concentration) x 100

Sample concentrations were corrected for procedural recoveries using the following equation:
Corrected concentration, mg/mL = Analysed concentration, mg/mL x 100 /R

Where
Ac = Peak response for test item in single standard.
As = Peak response for test item in sample chromatogram.
RF = Mean response factor of appropriate set of bracketing standard
V = Dilution volume (mL)
W = Sample weight (g)
R = Appropriate procedural recovery value
D = Density of sample (g/mL)
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - vehicle only
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males, 10 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The doses used in this study (0, 10, 30 and 125 mg/kg/day) were selected in conjunction with the Sponsor.

Dose levels were selected for this study, based on the results of a 14-day dose range finding study (Harlan Project No. 0142-0416) and a combined repeat dose toxicity study with production/developmental toxicity screening test repeated dose study (Harlan Project No. 0142-0417).

In the 14-day dose range finding study all animals given 250 mg/kg/day were sacrificed on Day 10 due to adverse clinical signs such as lethargy, hunched posture, dehydration and diarrhea and there was also significant body weight loss, increased water intake and reduced food consumption. After 14 days at 150 mg/kg/day there were no adverse clinical signs or body weight effects but the macroscopic investigations revealed increased liver weight and thickening of the non-glandular region of the stomach.

Based on these findings a high dose level of 125 mg/kg/day was selected for use on the reproduction/developmental toxicity screening test repeated dose study. In that study there were no adverse clinical signs and no effects on body weight or food consumption, however slightly increased liver and spleen weights were observed at macroscopic examination. At microscopic examination, acanthosis was seen in the fore-stomach of males and females treated at 125 mg/kg/day. Since the forestomach present in the rodent is not present in the human stomach and the functionally of the stomach was not compromised, these findings are not considered to be indicative of a risk to human health.

Consequently, 125 mg/kg/day was selected as the high dose level in this 90-day study. The intermediate and low dose levels of 30 and 10 mg/kg/day, respectively, were selected to allow evaluation of any dose related trends and to determine the no-observed-adverse-effect level.
Positive control:
None
Observations and examinations performed and frequency:
Serial Observations
Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Daily during the treatment period, detailed observations were recorded at the following times in relation to dose administration:

Daily during Week 1 of treatment Pre-dose observation. One to two hours after completion of dosing of all groups. As late as possible in the working day.

Daily from Week 2 of treatment Pre-dose observation. One to two hours after completion of dosing of all groups.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer who was unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction (e.g. approaches and/or sniffs probe)
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response (e.g. ear twitches/flattens or animal shakes its head)
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response (e.g. ear twitch only)
3 Normal response (e.g. obvious flinch or startle)
4 Exaggerated response (e.g. all feet off floor)

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response (e.g. turns around slowly or weak vocalization without moving away)
3 Normal response (e.g. jumps forward or turns around sharply, usually with vocalization)
4 Exaggerated response (e.g. excessive vocalization, body movement or aggression)

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor Activity
During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored. These data are retained in the study data but are not reported.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Water Consumption
Fluid intake was assessed by daily visual observation. No effect was observed; consequently quantitative measurements were not performed.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope (at the discretion of the examining veterinary surgeon a slit-lamp biomicroscope was also used) as follows:
Occasion Animals
Pretreatment All animals
Week 12 All animals of Groups 1 and 4
Week 13 All animals of Groups 2 and 3

Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and prior to dosing at the following occasion:
Occasion Animals
Week 13 All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

* Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected after overnight withdrawal of food and prior to dosing at the following occasion:
Occasion Animals
Week 13 All animals

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea*
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.
*Numerically equivalent to blood urea nitrogen (BUN)

Urine Collection
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasions:
Occasion Animals
Week 4 All animals
Week 13 All animals

The samples were collected and stored deep frozen (-60 to -90C) before dispatch. The samples were dispatched to the Responsible Scientist on dry ice.
The results of the analysis of the urine samples will be reported separately and are not part of this study.
Sacrifice and pathology:
Terminal Investigations

Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.

Necropsy
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Schedule Animals were killed following 13 weeks of treatment.

Sequence To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows:

Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities * * *
Adrenals * * * *
Aorta * * *
Brain (cerebellum, cerebrum, midbrain) * * * *
Cecum * * *
Colon * * *
Duodenum * * *
Epididymides * * * *
Esophagus * * *
Eyes * * *
Femur (femorotibial joint) * † †
Head * # #
Heart (including auricular and ventricular regions) * * * *
Ileum * * *
Jejunum * * *
Kidneys * * * *
Liver (section from two lobes) * * * *
Lungs (section from two major lobes including bronchi) * * *
Lymph nodes - mesenteric * * *
- left axillary * * *
Ovaries * * * *
Pancreas * * *
Pituitary * * *
Prostate * * *
Salivary glands - submandibular * † †
- sublingual * † †
- parotid * † †
Sciatic nerves * † †
Seminal vesicles * * *
Skin with mammary glands (inguinal area) * * *
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels) * * *
Spleen * * * *
Sternum (and bone marrow) * * *
Stomach * * *
Testes * * * *
Thymus * * * *
Thyroid with parathyroids * * *
Trachea * * *
Urinary bladder * * *
Uterus with cervix * * * *
Vagina * * *
* Organs weighed, samples fixed or sections examined microscopically.
# Not examined.
† Only one examined.
Animal ID retained

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at the end of the treatment period.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List All animals of Groups 1 and 4 killed at the end of the treatment period.
Abnormalities, eyes, liver, thyroid and stomach only All animals of Groups 2 and 3 killed at the end of the treatment period.
Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Other examinations:
Serial Observations
Clinical Observations
Signs are considered in two parts: detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’, and extended changes in condition, classified as ‘detailed physical examination and arena observations’.
Clinical observations are presented, providing detail of type of sign, day or week of occurrence and information on the duration of the sign applicable.
Body Weight
Group mean weight changes were calculated from the weight changes of individual animals.
Food Consumption
Group mean food consumptions and standard deviations for each period were derived from unrounded cage values. Overall mean food consumption values were calculated for each cage and the mean of these cage means were calculated for each group/sex.
Hematology, Peripheral Blood
The abbreviation used has the following meaning:
INS Insufficient sample

Blood Chemistry
Albumin to globulin ratio (A/G Ratio) was calculated as:
A/G Ratio = Albumin concentration
Total protein – albumin concentration

Blood urea nitrogen is numerically equal to, and is derived from urea since the results are expressed as mmol/L.
3.8.2 Terminal Investigations
Organ Weights
Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.
Pathology
The abbreviations used have the following meanings:
Lt Left
GALT Gut associated lymphoid tissue
Statistics:
Please refer to "Any other information on materials and methods incl. tables"
Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behaviour of animals receiving 10 or 30 mg/kg/day and the behaviour of animals receiving 125 mg/kg/day were unaffected by treatment and no deaths occurred during the study.
Mortality:
no mortality observed
Description (incidence):
No deaths occured during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The overall weight gain of males receiving 30 or 125 mg/kg/day were slightly lower than the controls (5 and 6% reduction, respectively) but statistical significance was not attained and, consequently, these trends were attributed to normal biological variation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
The ophthalmic examination in Week 12 indicated the presence of opacification/cataract in the lens of seven males and seven females receiving 125 mg/kg/day. In the majority of affected animals this presented as slight opacification/cataract on the posterior capsule but in two males and two females was a seen as a total nuclear cataract that was bilateral in one male and one female and unilateral in one male and one female. The presence of the total nuclear cataract obscured examination of the vitreous and fundus but in animals where these structures could be assessed there was no evidence for any treatment-related change.
In view of the presence of the lenticular findings, the ophthalmoscopic examination was extended to males and females treated at 10 or 30 mg/kg/day (examination performed in Week 13), there were no treatment-related findings at these doses.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematology, Peripheral Blood
The haematological examination in Week 13 indicated low haematocrit, haemoglobin concentration and mean cell volume and a marginal increase of red cell distribution width in females receiving 125 mg/kg/day. There was also a small reduction of mean cell volume in females receiving 10 or 30 mg/kg/day but this was unlikely to be of any toxicological significance in the absence of any change in the erythrocyte indices at these doses. With the exception of low mean cell volume, these trends were not evident in males. In males there was a marginal increase in erythrocyte count and decreased mean cell volume at 30 or 125 mg/kg/day and low mean cell haemoglobin at 125 mg/kg/day but these were considered of no toxicological significance, particularly as haemoglobin concentration was unaffected.
Males receiving 30 mg/kg/day and males and females receiving 125 mg/kg/day showed an increase, compared to controls, of neutrophil count, with the extent of the effect in males being dose-related, and lymphocyte counts were high in females receiving 125 mg/kg/day. In addition, at 125 mg/kg/day, there were also increases of eosinophil count in males, basophil and large unstained cell count in females and monocyte count in both sexes but the these differences from controls were minor. As a consequence of these findings, the total leucocyte counts of animals receiving 125 mg/kg/day were higher than controls.

All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the slightly but statistically significantly shorter activated partial thromboplastin time in males receiving 125 mg/kg/day since reduced clotting times are considered of no toxicological significance, prothrombin times were unaffected and there was no similar finding in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood Chemistry
The biochemical examination of the blood plasma in Week 13 indicated, when compared to controls, high urea concentrations in males and females receiving 125 mg/kg/day which, in females, associated with low plasma creatinine concentration.

The plasma glucose concentrations of males receiving 125 mg/kg/day were slightly higher than controls, but there was no similar finding in females.

In females treated at 125 mg/kg/day there was an increase of total plasma cholesterol concentration but there was no effect on triglyceride concentration and males were unaffected.

Calcium concentrations were increased, compared to controls, in females treated at 30 or 125 mg/kg/day but there was no similar finding in males.

Total protein was increased, compared to controls, in females treated at 125 mg/kg/day. This was attributed to an increase in both the albumin and globulin fractions and led to a decrease of the albumin to globulin ratio. There was also a small reduction of the albumin to globulin ratios of females receiving 30 mg/kg/day. Males were unaffected.

All other inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the high aspartate amino transferase activities in males receiving 10 mg/kg/day since this was due to high values in four animals (with values >100 U/L) and there was no similar finding at higher doses. They also included the statistically significantly low total bilirubin concentrations in females receiving 125 mg/kg/day since none of the individual values was abnormal and reduced bilirubin concentrations are of no toxicological importance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength were unaffected by treatment.

Motor Activity
Motor activity was unaffected by treatment.

Statistical significance was attained in the males receiving 10 mg/kg/day for an increase of low (cage-floor activity) beam breaks at the 6 minute interval and for the increase of high (rearing) beam breaks at the 24 minute interval in females receiving 125 mg/kg/day. These were transient variations that were confined to one sex and. in the absence of any alteration of total beam breaks, were attributed to normal variation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of organ weights after 13 weeks of treatment indicated, when compared to controls, high body weight-adjusted adrenal gland weight in males given 125 mg/kg/day, high body weight-adjusted kidney weight in males and females given 125 mg/kg/day, and high body weight-adjusted liver weight in females given 30 mg/kg/day and in males and females given 125 mg/kg/day.

All other inter-group differences from control were minor, lacked dose-relationship or (where applicable) were confined to one sex and were therefore attributed to normal biological variation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macropathology
The macroscopic examination performed after 13 weeks of treatment revealed the following changes in the stomach and eyes related to the test item.

Stomach
Thickened nonglandular stomach mucosa was seen in all males treated at 30 or 125 mg/kg/day and in females treated at 10 (1 animal), 30 (7 animals) or 125 (all animals) mg/kg/day.

Eye
Unilateral or bilateral lens opacity was observed in a few animals treated at 125 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings
Changes related to treatment with Ethanol, 2,2'-iminobis-, NC12-18-alkyl derives were seen in stomach (nonglandular stomach), liver, thyroid glands and eyes.

Nonglandular stomach
Epithelial hyperplasia (acanthosis) and hyperkeratosis was seen in animals treated with 30 or 125 mg/kg/day, with the extent of this finding being dose-related. A female given 10 mg/kg/day was also affected, to a minimal extent.

Thyroid glands
Hypertrophy of the follicular cells was observed in both sexes treated at 125 mg/kg/day.

Liver
Increased rarefaction of the hepatocytes was seen in both sexes treated at 30 or 125 mg/kg/day, with the incidence being higher in females than in males.

Eyes
Lens degeneration was observed in four males and three females given 125 mg/kg/day.

Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
ophthalmological examination
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
ophthalmological examination
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
eye
Organ:
lens
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

 Macropathology

Macroscopic examination performed after 13 weeks of treatment revealed the following changes in the stomach and eyes related to the test item.

Stomach

Thickened nonglandular stomach mucosa was seen in all males treated at 30 or 125mg/kg/dayand in females treated at 10 (1 animal), 30 (7 animals) or 125 (all animals)mg/kg/day.

Summary of findings in the stomach for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Thickened – Nonglandular Mucosa

0

0

10

10

0

1

7

10

Number of animals examined

10

10

10

10

10

10

10

10

Eye

Unilateral or bilateral lens opacity was observed in a few animals treated at 125 mg/kg/day.

Summary of findings in the eye for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Opaque

0

0

0

3

0

0

0

2

Number of animals examined

10

10

10

10

10

10

10

10

The incidence and distribution of all other findings were considered to be unrelated to treatment.

  Histopathology

Treatment-related findings

Changes related to treatment with Ethanol, 2,2'-iminobis-, NC12-18-alkyl derives were seen in stomach (nonglandular stomach), liver, thyroid glands and eyes.

Nonglandular stomach

Epithelial hyperplasia (acanthosis) and hyperkeratosis was seen in animals treated with 30 or 125 mg/kg/day, with the extent of this finding being dose-related. A female given 10 mg/kg/day was also affected, to a minimal extent.

Summary of treatment related findings in the nonglandular stomach for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Epithelial hyperplasia (acanthosis)

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

1

2

0

Slight

0

0

3

1

0

1

7

1

Moderate

0

0

6

6

0

0

0

9

Marked

0

0

0

2

0

0

0

0

Total

0

0

10

10

0

2

9

10

Hyperkeratosis

 

 

 

 

 

 

 

 

Minimal

0

0

1

1

0

1

4

0

Slight

0

0

6

1

0

0

5

1

Moderate

0

0

2

8

0

0

0

9

Total

0

0

9

10

0

1

9

10

Number of tissues examined

10

10

10

10

10

10

10

10

Thyroid glands

Hypertrophy of the follicular cells was observed in both sexes treated at 125 mg/kg/day.

Summary of treatment related findings in the thyroid glands for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Hypertrophy, Follicular cells

 

 

 

 

 

 

 

 

Minimal

0

0

0

4

0

0

0

5

Slight

0

0

0

3

0

0

0

2

Total

0

0

0

7

0

0

0

7

Number of tissues examined

10

10

10

10

10

10

10

10

Liver

Increased rarefaction of the hepatocytes was seen in both sexes treated at30 or125 mg/kg/day, with the incidence being higher in females than in males.

Summary of treatment related findings in the liver for animals killed after 13 weeks of treatment

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Rarefaction, Increased

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

0

0

1

7

Slight

0

0

0

1

0

0

0

0

Total

0

0

0

3

0

0

1

7

Number of tissues examined

10

10

10

10

10

10

10

10

Eyes

Lens degeneration was observed in four males and three females given125 mg/kg/day.

 

Summary of treatment related findings in the eyes for animals killed after 13 weeks of treatment

 

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

10

30

125

0

10

30

125

Degeneration, Lens

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

0

0

1

Slight

0

0

0

1

0

0

0

2

Moderate

0

0

0

2

0

0

0

0

Total

0

0

0

4

0

0

0

3

Number of tissues examined

10

10

10

10

10

10

10

10

Incidental findings

The incidence and distribution of all other findings were considered to be incidental and unrelated to treatment.

Attached

TABLES  

TABLE 1 JH45BW 90-day rat study Detailed physical examination and arena observations - group distribution of observations      

                               

TABLE 2 JH45BW 90 day rat study Sensory reactivity observations and grip strength - summary of findings during Week 12 of treatment   

       

TABLE 3 JH45BW 90 day rat study Motor activity - group mean scores (beam breaks) during Week 12 of treatment  

                   

TABLE 4 JH45BW 90 day rat study Body weight - group mean values (g)      

                            

TABLE 5 JH45BW 90 day rat study Food consumption - group mean values (g_animal_week)        

    

TABLE 6 JH45BW 90 day rat study Ophthalmic examination - group distribution of observations before commencement of treatment and during Week 12 or 13

TABLE 7 JH45BW 90 day rat study Hematology - group mean values during Week 13 of treatment

TABLE 8 JH45BW 90 day rat study Blood chemistry - group mean values during Week 13 of treatment

TABLE 9 JH45BW 90 day rat study Organ weights - group mean absolute and adjusted values (g) for animals killed after 13 weeks of treatment

TABLE 10 JH45BW 90 day rat study Macropathology - group distribution of findings for animals after 13 weeks of treatment

TABLE 11 JH45BW 90 day rat study Histopathology - group distribution of findings for animals after 13 weeks of treatment                                                          

 

FIGURES   

                                                                                                                                 

FIGURE 1 JH45BW 90 day rat study Motor activity - group mean scores (beam breaks) for males and females during Week 12 of treatment            

                                     

FIGURE 2JH45BW 90 day rat study Body weight - group mean values (g) for males and females

APPENDIX

Results Envigo Study Number JH45BW 90 day rat study Formulation Analysis results

 

Conclusions:
It is concluded that oral administration of Ethanol, 2,2'-iminobis , NC12-18-alkyl derives (a substance used in industry) to Han Wistar rats for 13 weeks caused adverse findings in the eyes at 125 mg/kg/day (lenticular opacification/cataract and degeneration) and stomach at 30 and 125 mg/kg/day (epithelial hyperplasia (acanthosis) and hyperkeratosis). The findings in the nonglandular stomach are considered to be due to local irritant effects of the test formulations. There were also rodent-specific findings in the thyroid glands (follicular cell hypertrophy), that was likely secondary to induction of liver enzymes, and a minor and non adverse effect on hepatocellular glycogen (increased rarefaction). In view of the presence of adverse findings in the eyes at 125 mg/kg/day the systemic no-observed-adverse-effect level (NOAEL) in this study was considered to be 30 mg/kg/day.
Executive summary:

 Summary

The purpose of this study was to assess the systemic toxic potential of Ethanol, 2,2'‑iminobis‑, NC12-18-alkyl derives (a substance used in industry) when administered orally, by gavage, to Han Wistar rats for 13 weeks. Three groups, each comprising ten males and ten females, received the test material at doses of 10, 30 or 125 mg/kg/day and a similarly constituted control group received the vehicle (arachis oil)at the same volume dose. 

During the study, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

Results

The general behavior, sensory reactivity responses, grip strength and motor activity were not affected by treatment, no deaths occurred during this study and there was no effect of treatment on body weight gain or on food and water consumption.

Opaque eyes were observed from Week 9 in three males and two females receiving 125 mg/kg/day. In most cases this was initially a unilateral finding that rapidly became bilateral as the treatment period progressed. Animals receiving 10 or 30 mg/kg/day were unaffected.

The ophthalmic examination in Week 12 indicated the presence of opacification/cataract in the lens of seven males and seven females receiving 125 mg/kg/day, located predominantly on the posterior capsule but in two males and two females there was a total nuclear cataract.

The haematological examination in Week 13 indicated low haematocrit, haemoglobin concentration and mean cell volume and a marginal increase of red cell distribution width in females receiving 125 mg/kg/day, with low mean cell volume also occurring in males at this dose. Treatment-related alterations of leucocyte numbers included increased neutrophil count in males receiving 30 mg/kg/day and males and females receiving 125 mg/kg/day, increased lymphocyte counts in females receiving 125 mg/kg/day and minor increases at 125 mg/kg/day of eosinophil count in males, basophil and large unstained cell count in females and monocyte count in both sexes, leading to increased total leucocyte counts in animals receiving 125 mg/kg/day.

Biochemical changes in the blood plasma in Week 13 comprised: high urea concentrations in males and females receiving 125 mg/kg/day; low creatinine and high total cholesterol concentrations in females receiving 125 mg/kg/day; high glucose concentrations in males receiving 125 mg/kg/day; increased calcium concentrations in females treated at 30 or 125 mg/kg/day; high total protein concentration in females receiving 125 mg/kg/day, which was attributed to an increase in both the albumin and globulin fractions and led to a decrease of the albumin to globulin ratio. 

Organ weight analysis after 13 weeks of treatment indicated high adrenal gland weight in males given 125 mg/kg/day, high kidney weight in males and females given 125 mg/kg/day and high liver weight in females given 30 mg/kg/day and in males and females given 125 mg/kg/day.

The macroscopic examination performed after 13 weeks of treatment revealed thickening of the nonglandular mucosa of the stomach at 30 and 125 mg/kg/day, with one female at 10 mg/kg/day being similarly affected, and unilateral or bilateral lens opacity in three males and two females treated at 125 mg/kg/day.

Histopathological findings that were attributed to treatment occurred in the nonglandular stomach (epithelial hyperplasia (acanthosis) and hyperkeratosis at 10 (one female, minimal), 30 and 125 mg/kg/day, with the extent of this finding being dose-related), liver (increased hepatocyte rarefaction in animals, particularly females, given 125 mg/kg/day), thyroid glands (follicular cell hypertrophy at 125 mg/kg/day) and eyes (lenticular degeneration infour males and three females given125 mg/kg/day).

Conclusion

It is concluded that oral administration of Ethanol, 2,2'-iminobis‑, NC12-18-alkyl derives (a substance used in industry) to Han Wistar rats for 13 weeks caused adverse findings in the eyes at 125 mg/kg/day (lenticular opacification/cataract and degeneration) and stomach at 30 and 125 mg/kg/day (epithelial hyperplasia (acanthosis) and hyperkeratosis). The findings in the nonglandular stomach are considered to be due to local irritant effects of the test formulations. There were also rodent-specific findings in the thyroid glands (follicular cell hypertrophy), that was likely secondary to induction of liver enzymes, and a minor and non-adverse effect on hepatocellular glycogen (increased rarefaction). In view of the presence of adverse findings in the eyes at 125 mg/kg/day the systemic no-observed-adverse-effect level (NOAEL) in this study was considered to be 30 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Very good
System:
eye
Organ:
lens

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The mode of action for the effects in the eyes is not known, effects on the eyes were not seen in a 90 day study at a higher 150mg/kg/day dose level in a related substance with a longer carbon chain distribution C16-C18, 2,2'-(Octadec-9 -enylimino)bisethanol CAS No 25307 -17 -9. The relevance of this effect for humans is not known. However, the clear no effects level at 30mg/kg and the unlikely human exposure by ingestion of amounts in excess of the oral DNEL means that the risk to human will be very low.

Additional information

In a 14 day dose ranging study The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31- 9) to rats by gavage for a period of up to fourteen consecutive days at dose levels of 250, 150 and 75 mg/kg/day resulted in significant toxicity at 250 mg/kg/day. Rats dosed at this level were terminated after 10days due to their poor clinical condition.

 

Treatment-related effects including increased liver weights and macroscopic gastric changes were also evident at 150 and 75 mg/kg/day; therefore a ‘No Observed Effect Level’ (NOEL) was not established at the dose levels employed in the fourteen day range-finding phase.

 

The information from the 14 day study was used to select the dose levels for an OECD guideline 422 (Gavage)Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test(adopted 22 March 1996).

Wistar Han™:HsdRccHan™:WIST strain rat for up to forty-five days(including a two week maturation phase, pairing, gestation and early lactation),at dose levels of 10, 30 and 125 mg/kg/day. A control group was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males, were treated with the high dose (125 mg/kg/day) or the vehicle alone for forty-two days and then maintained without treatment for a further fourteen days

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected males and females from each dose group.

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

 

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4post partum.

 

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum.  All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

The oral administration of Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9) to rats by gavage, at dose levels of 125, 30 and 10 mg/kg/day, resulted in treatment-related effects at 125 and 30 mg/kg/day. These effects consisted of a localised irritant effect in the stomach tract, and almost complete regression was evident following the treatment-free period. This change may be considered to be an adverse event and of importance when considering the impact on reproductive performance. Based upon the histopathological changes observed in the stomach, which are considered to be a local response to the administration by gavage of the corrosive test substance, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity, was considered to be 30 mg/kg/day.

As 2, 2’-(C12-18 evennumbered alkyl imino) diethanol is a high tonnage Annex X substance, ECHA required an OECD408, 90-day (13 week) study in rats. This studyinvolved gavage dosing Han Wistar rats for 13 weeks, in this study the test substance was referred to as Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derives. Three groups, each comprising ten males and ten females, received the test material at doses of 10, 30 or 125 mg/kg/day and a similarly constituted control group received the vehicle (arachis oil) at the same volume dose.

 

During the study, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption,

ophthalmic examination, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

 

The general behaviour, sensory reactivity responses, grip strength and motor activity were not affected by treatment, no deaths occurred during this study and there was no effect of

treatment on body weight gain or on food and water consumption.

 

Opaque eyes were observed from Week 9 in three males and two females receiving 125 mg/kg/day. In most cases this was initially a unilateral finding that rapidly became

bilateral as the treatment period progressed. Animals receiving 10 or 30 mg/kg/day were unaffected.

 

The ophthalmic examination in Week 12 indicated the presence of opacification/cataract in the lens of seven males and seven females receiving 125 mg/kg/day, located predominantly

on the posterior capsule but in two males and two females there was a total nuclear cataract.

Oral administration of Ethanol, 2,2'-iminobis-, N-C12-18-alkyl derives to Han Wistar rats for 13 weeks caused adverse findings in the eyes at 125 mg/kg/day (lenticular opacification/cataract and degeneration) and stomach at 30 and 125 mg/kg/day (epithelial hyperplasia (acanthosis) and hyperkeratosis). The findings in the nonglandular stomach are considered to be due to local irritant effects of the test formulations. There were also rodent-specific findings in the thyroid glands (follicular cell hypertrophy), that was likely secondary to induction of liver enzymes, and a minor and nonadverse effect on hepatocellular glycogen (increased rarefaction).

 

In view of the presence of adverse findings in the eyes at 125 mg/kg/day the systemic no-observed-adverse-effect level (NOAEL) in this study was considered to be 30 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

The results detailed above from the OECD408 90-day study in rats are consistent in terms of the NOAEL for systemic toxicity with the OECD422 study where both have a NOAEL for systemic toxicity of 30 mg/kg bw/day. Different test substance names have been used in these studies for the substance registered as  Ethanol, 2,2-iminobis-, N-C12-18-alkyl derivs, it is also referred to asEthanol, 2, 2’-iminobis-, N-coco alkyl derives or 2, 2’-(C12-18 evennumbered alkyl imino) diethanol all three are the same substance.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

The low vapour pressure of the substance means inhalation is not considered to be relevant route of exposure so not testing is required.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

The corrosive properties of this substance mean the repeated dose dermal studies are not scientifically justified due to concerns for animal welfare.  

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

Local effects would be expected to be irritation/corrosion which are local concentration rather than dose dependent.  Specific testing is not scientifically justified due to concerns for animal welfare.  The classification as corrosive will ensure dermal exposure is well controlled.

Repeated dose toxicity: via oral route - systemic effects (target organ): eyes:lenticular opacification/cataract and degeneration. digestive: stomachepithelial hyperplasia (acanthosis) and hyperkeratosis.

Justification for classification or non-classification

The NOAEL in the OECD 408 90-day study is 30mg/kg/day, systemic effects were only seen in the 125mg/kg/day group.

 

The EU CLP (GHS) criteria for classification for Specific Target Organ Toxicity (STOT) are based on data from a 90-day study, for Category 2 the range for such effects is 10-<100 mg/kg/day. Where the data are

As the highest dose in this study was 125 mg/kg/day and the NOAEL was for the middle dose of 30 mg/kg/day, it could potentially require a classification for category 2 for STOT. Findings in

the 125mg/kg/day group were mainly in the eyes wherelenticular opacification/cataract and degenerationwere seen. The other histopathological findings were of acanthosis and hyperkeatosis

in the fore stomach of the rats. While this was considered an adverse effect of the test substance

it was a local effect due to gavage dosing of a severely irritant/corrosive test substance into the stomach and not a systemic toxic effect. Also, the fore stomach of the rat is non-glandular and therefore it is not representative of the human stomach which is does not have a non-glandular region. There were also rodent-specific findings in the thyroid glands (slight/mild follicular cell hypertrophy), that was likely secondary to induction of liver enzymes, and a minor and none adverse effect on hepatocellular glycogen (increased rarefaction).

 

The only histopathological effects seen that could be considered as possibly requiring a classification for Specific Target organ Toxicity(STOT) were of cataracts which were only seen in 3 males and 2 females dosed with 125 mg/kg/day. The upper limit for classification as STOT category 2 in a 90-day study is 100mg/kg. There were no indications either from ophthalmology or histopathology of any cataracts or other adverse effects in the eyes at the 30mg/kg middle dose.  The effects in the eyes were seen at a dose level higher than 100mg/kg/day with no indications of any effects on the eyes at the middle dose. Based on this I do not consider it necessary to classify for STOT category 2.