Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
No data available. An EOGRTS is proposed.
Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study planned
Study period:
2016-2018, depending on ECHA's timeline for permission. Minimum 30 months from Final Desicion
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Amines, polyethylenepoly-, triethylenetetramine fraction. CAS 90640-67-8

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no GLP-compliant studies available on reproductiv toxicity with the registered substance.
- Available non-GLP studies: There are no non-GLP studies available on reproductive toxicity with the registered substance.
- Historical human data: There are no appropriate historical human data available addressing the endpoint reproductive toxicity.
- (Q)SAR: (Q)SAR tools sufficiently addressing the endpoint reproductive toxicity are currently not available.
- In vitro methods: No validated or regulatory accepted alternative methods are available for replacing animal testing with respect to reproductive toxicity.
- Weight of evidence: There are no studies available on repeated dose toxicity with the registered substance which could be used in a weight of evidence approach.
- Grouping and read-across: There are no read-across data available on toxicity to reproduction

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- There is no data available on reproductive toxicity with Amines, polyethylenepoly-, triethylenetetramine fraction (CAS 90640-67-8). Validated or regulatory accepted alternative methods are not available for replacing animal testing with respect to reproductive toxicity. Furthermore, although appropriate read-across substances are available (see read across justification) for none of these substances a reproductive toxicity study is available so far.
In order to fulfil the standard information requirements, a GLP-compliant reproductive toxicity study in the rat via the oral route following OECD 443 was proposed, according to Annex IX, Column I, 8.7.2.

Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
Testing Proposal for an EOGRTS without extension of Cohort 1B, without Cohorts 2A and 2B, and with Cohort 3.

Justification:

Pre-mating exposure period of 10 weeks as recommended in the draft Guideline.

Dose level setting: a top dose level will be selected which leads to minimal signs of systemic toxicity in pregnant animals, as per the draft Guideline. From earlier developmental toxicity studies with analogues, it is known that pregnant female animals may be more sensitive to systemic ethylene amine toxicity than not-pregnant females or males, therefore a range-finding study in pregnant animals will be necessary.

Extension of Cohort 1B is deemed not to be necessary since none of the triggers from Column II of Annex X are applicable.

Inclusion of Cohorts 2A / 2B is not deemed necessary, since no signs of neurotoxicity were observed in any study with the test material or its analogues. In view of the very high water solubility and very low Kow, penetration in fat-rich tissue, c.q. the developing brain and other nervous tissues, is not to be expected.

Cohort 3 is not deemed necessary since, apart from the known skin sensitisation properties, no effects on the immune system are known of TETA and other ethylene amines.
Species:
rat
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
plus neutralisation with HCl to pH < 9
Details on exposure:
exposure by gavage to ensure proper dosing, neutralisation to pH < 9 will be required because of the corrosive nature of the substance.
Details on mating procedure:
according to OECD protocol
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
according to OECD protocol
Duration of treatment / exposure:
according to OECD protocol
Frequency of treatment:
according to OECD protocol
Details on study schedule:
according to OECD protocol
No. of animals per sex per dose:
according to OECD protocol
Parental animals: Observations and examinations:
according to OECD protocol
Oestrous cyclicity (parental animals):
according to OECD protocol
Sperm parameters (parental animals):
according to OECD protocol
Litter observations:
according to OECD protocol
Postmortem examinations (parental animals):
according to OECD protocol
Postmortem examinations (offspring):
according to OECD protocol
Statistics:
to be determined.
Reproductive indices:
according to OECD protocol
Offspring viability indices:
according to OECD protocol
Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No studies were located that could provide information on effects to fertility. An EOGRTS is proposed.


Effects on developmental toxicity

Description of key information

OECD 414 (rat, oral, reliability 2): NOAEL maternal toxicity = 750 mg/kg bw/day; NOAEL developmental = 750 mg/kg bw/day (highest dose tested)

similar to OECD 414 (rabbit, dermal, reliability 2): NOAEL maternal toxicity = 50 mg/kg bw/day; NOAEL developmental = 125 mg/kg bw/day (highest dose tested)

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-01-03 To: 1984-12-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study was conducted according to OECD Guideline 414 from that time, using exposure from gestation day 6 to day 15 . However, the current guideline requires exposure from at least day 6 up to and including the day before giving birth. No data on GLP and no data on the analytical verification of the dosing solutions.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
the current guideline requires exposure from at least gestation day 6 up to and including the day before planned delivery; not tested up to 1000 mg/kg bw/day
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: albino, Sprague-Dawley-derived [(TIf:RAIf (SPF)]
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from a closed breeding colony (CIBA-GEIGY, WST)
- Age at study initiation: 2 months old
- Weight at study initiation: 190-200 g
- Housing: Makrolon cages equipped with a wire mesh top and water bottles, saw dust (granular form) serving as bedding material, housed 4 per cage throughout the experiment.
- Diet: Certified standard cube diet (NAFAG No. 890), ad libitum
- Water: Tap water, ad litibum
- Acclimation period: Acclimatization took place in the period of time between allocation to the corresponding group on day 0 and the first treatment on day 6 post coitum.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21C ± 2C
- Humidity (%): 55% ± 10%
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
other: Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was prepared fresh daily dissolved in distilled water by magnetic stirrer, and administered at a rate of 10 ml/kg of body weight by oral intubation.

Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male/3 female
- Length of cohabitation: Overnight
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
Day 6 until Day 15 of pregnancy
Frequency of treatment:
Daily
Duration of test:
Duration of exposure = 10 days, Duration of test = 21 days (copulation through day 21 of pregnancy)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
325 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 dams/dose group in preliminary study and 24 dams/dose per group in main study
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: In order to determine the dose levels for the main study, a preliminary experiment was carried out on 12 fertilized rats each for the vehicle control and the dose groups (350 and 700 mg/kg). The test material was dissolved in distilled water and administered orally by intubation from day 6 until day 15 of pregnancy, inclusive. Treatment at these dose levels caused a decrease of food consumption in the mother animals of the 700 mg/kg group. No adverse effects were recorded for the progeny at sacrifice shortly before term in comparison with the vehicle control. On the basis of the foregoing results the doses for the main study were selected at 0, 75, 325 and 750 mg/kg of body weight. The test material was again mixed with distilled water and administered once daily by the oral route from day 6 until day 15 of pregnancy, inclusive.
- Rationale for animal assignment (if not random): The mated females were identified by color code, allocated to experimental and control groups (24 animals per group) by using a randomization table
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: General bodily conditions and symptoms

BODY WEIGHT: Yes
- Time schedule for examinations: Daily


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Days 6, 11, 16, and 21 of pregnancy


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: ovaries and uterus (including: mucosa and contents, including amniotic fluid and placentae as well as abortions and resorption sites)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [one third per litter]
- Skeletal examinations: Yes: [two thirds per litter]
Statistics:
When feasible, statistical evaluation of data was performed. Progency abnormalities were evaluated by Student's t-test (one-tailed), Embryonic and/or foetal deaths and male to female ratios were evaluated by Chi square test with Yates correction, Average weight of live foetuses were evaluated with Student's t-test (one-tail).
Historical control data:
The current study used concurrent controls and historical data to compare the results.
Details on maternal toxic effects:
Maternal toxic effects: yes

Details on maternal toxic effects:
With regard to the vehicle control, food intake was slightly depressed in all dose groups during the second part of the treatment period. Body-weight gain appeared to be somewhat reduced in the experimental groups; however when corrected body weights were evaluated no differences were observed.
In the vehicle control a spontaneous litter (i.e. parturition of normally developed foetuses one or two days prior to expected delivery) was observed for one female on day 20 p.c. An early birth of the whole litter (i.e. shortly before sacrifice on day 21) was recorded for one female of the mid dose group of the main study. These animals were not taken into further consideration of data. Early birth of 5 foetuses was also noted for one female of the low-dose group in the main study, 3 of these foetuses were cannibalized, the remaining were assigned at random to a uterine location and processed as usual.
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects up to the highest dose tested
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
The average body-weight of the live foetuses of the low and mid dose groups from the main study were found to be slightly enhanced in comparison with the vehicle control. This, however, was not thought to be of biological relevance; a more significant increase was recorded for the high-dose group in the main study (Student's t test, one-tailed, observed p < 0.01). The gross inspection of the live foetuses did not reveal any finding either in the experimental groups or in the vehicle control. Carrying out the slicing technique for "visceral" examination, internal hydrocephaly (unilateral) was found in one foetus of the low-dose group in the main study. Encephalocele occurred in one foetus of the vehicle control. The skeletal assessment revealed instances of irregular sternebral ossification in the dose groups and in the vehicle control. Concerning the status of skeletal maturation of the foetuses shortly before term, apart from a slightly raised number of still unossified calcanei in the high-dose group in the main study, no delay of ossification could be found for the experimental groups in comparison with the vehicle control; in view of the ranges ascertained for the "historical" control, however, this finding was not thought to be of experimental significance.
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Developmental effects observed:
no

The pregnancy rates expressed as average number of implantation sites per female were comparable for all groups. The numbers of embryonic and/or foetal deaths (resorptions) were not increased at either dose (Chi2-test, Yates' correction, observed p>0.05) in the main study. The male to female ratios of the foetuses were also comparable for all groups (Chi2-test, Yates' correction, observed p>=0.05) in the main study.

Conclusions:
The test substance was devoid of any embryotoxic activity and did not reveal teratogenic potency in the rat under the present experimental conditions.
Executive summary:

Timed-pregnant Sprague Dawley rats were treated with the test substance by daily oral gavage on gestational days (gd) 6 up to and including day 15. Groups of 24 females each received the test substance formulated in distilled water applied at a volume of 10 ml/kg bw at dosages of 0, 75, 325 or 750 mg/kg bw per day. With regard to the vehicle control, food intake was slightly depressed in all dose groups during the second part of the treatment period. Body-weight gain appeared to be somewhat reduced in the experimental groups; however when corrected body weights were evaluated no differences were observed. No other toxic signs were observed in the mothers. A significant increase in the average body weight of the live foetuses was recorded for the high-dose group in the main study. The gross inspection of the live foetuses did not reveal any finding either in the experimental groups or in the vehicle control. The skeletal assessment revealed instances of irregular sternebral ossification in the dose groups and in the vehicle control. Concerning the status of skeletal maturation of the foetuses shortly before term, apart from a slightly raised number of still unossified calcanei in the high-dose group in the main study, no delay of ossification could be found for the experimental groups in comparison with the vehicle control.

Thus, under the conditions of this study, the test substance produced no maternal toxicity except for slightly reduced food intake and no developmental toxicity at any dosages employed.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May- June 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
- The animals were treated from gestational days 6 through 18
- No justification for the exposure route or duration was given, six hours per day via the skin.
- The body weights of the animals were not determined every 3 days.
- Only half of the pups was examined for skeletal abnormalities, it should be all pups for non-rodents.
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazleton Dutchland Laboratories, Inc., Denver
- Age at study initiation: 5-5.5 months old
- Weight at study initiation: 2.5-3.5 kg
- Fasting period before study: Not applicable
- Housing: The rabbits were individually housed in stainless steel wire-mesh cages (46 cm x 61 cm x 36 cm high)
- Diet (e.g. ad libitum): food (Agway Prolab@ Certified Rabbit Food@, Batch Nos. 7085 SM2, 7055 SM2 and 7098 SM2, Agway, Inc., St. Marys, OH) available ad libitum
- Water (e.g. ad libitum): water (Municipal Authority of Westmoreland County, Greensburg, PA) available ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature
- Humidity (%): was recorded but not reported
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: May 24 through June 8, 1987
Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 5 x 5 inch ± 12.7 x 12.7 cm
- % coverage: ± 10 % (for rabbits 12 x 14 cm is 10%)
- Type of wrap if used: The test material was applied under a 4 x 4 inch sterile gauze square (Johnson & Johnson Products, Inc., New Brunswick, NJ). The volume applied remained constant at 2.0 ml for all animals and was not corrected for individual animal body weights during the dosing period to maintain the dose volume constant. A Lycra-Spandex jacket with velcro closures, with a 5 x 5 inch square of polyethelene film attached (corresponding to the location of the dosing site) was used to occlude the dosing site. The rabbit's forelimbs were placed through the jacket's "armholes" and the jacket fastened in the back by the velcro closures. Each jacket was identified with the rabbit's unique identification number and was used only for that female throughout the dosing period. During the six-hour exposure period, the rabbits were checked periodically to see if the gauze and jacket were still in place. If not in place, the jacket and gauze were reapplied.
- Time intervals for shavings or clipplings: one to two days before the start of the treatment and any time throughout treatment as deemed necessary due to rapid hair growth.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: After six hours of exposure, the gauze and jacket were gently removed and the application site was wiped gently with a gauze strip dampened with warm water and blotted dry with a disposable wiper.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A 2.0 ml aliquot of the appropriate dosing solution was drawn up in a 3.0 cc syringe.
- Concentration (if solution): The dosing solutions were 0.0 (vehicle control), 10.0, 100.0 and 250.0 mg/ml for concentrations of 0.0, 5.0, 50.0 and
125.0 mg/kg/day (at 0.5 ml/kg and 2.0 ml/animal), respectively.
- Constant volume or concentration used: constant volume
- For solids, paste formed: not applicable


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Amount(s) applied (volume or weight with unit): see above
- Concentration (if solution): see above
- Lot/batch no. (if required): not applicable
- Purity: not applicable

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A standard stock solution was prepared of 1.0 mg/ml test chemical in deionized (Millipore@) water. Standards were prepared from 200 to 700 ng/µl by dilution with deionized water. A 1.0 ml aliquot of each dosing solution was diluted with deionized water to yield concentrations of 400 to 600 ng/µl. The diluted standard and dosing solutions were derivatized in saturated sodium bicarbonate and 70 mg/ml dansyl chloride solution in acetone and heated to 37C for one hour. Then toluene was added and the solutions centrifuged. An aliquot of the organic phase (20 µl) was then injected into a Waters' High Pressure Liquid Chromatograph with a Model 450 variable wavelength detector. The flow rate was 2 ml/min and the wavelength was 254 nm. The dosing formulations were homogeneous and stable for at least 21 days. Therefore, they were prepared once prior to the onset of the dosing period and analyzed at the time of preparation.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 3 days max.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data, rabbits were mated consecutively to two males (if possible): no data.
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: no data
- Any other deviations from standard protocol: no data
Duration of treatment / exposure:
gestational days 6 through 18
Frequency of treatment:
daily for 6 hours
Duration of test:
through gestational day 29
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range finding study
- Rationale for animal assignment (if not random): no data
Maternal examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All females were examined daily for clinical signs of toxicity and were observed twice daily during the dosing period (pre- and post-dosing) and once daily in the post-dosing period (gd 19-29) for any skin irritation, erythema, edema or eschar formation

BODY WEIGHT: Yes
- Time schedule for examinations: All females on study were weighed on gd 0, 6 (prior to onset of dosing), 12, 15, 18 (during the dosing period), and 29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
The maternal body cavities were opened by midline thoracolaparotomy. The gravid uterus, ovaries (including corpora lutea), cervix, vagina, and abdominal and thoracic organs and cavities were examined grossly. Maternal liver, kidney (2), lung (2) and uterine weights were determined. Kidneys were bisected (left longitudinally, right transversely) and liver sections were cut and fixed in buffered neutral 10% formalin for possible subsequent histopathologic examination. One square inch of skin was removed from the application site and fixed in buffered neutral 10% formalin, Maternal urine was removed from the urinary bladder by needle and syringe for subsequent analysis for copper content.
OTHER: On gd 29, each rabbit was restrained and approximately 3.0 ml of blood was removed by cardiac puncture into tubes without anticoagulant (Vacutainex-63 Brand, Becton-Dickinson, Rutherford, NJ) for subsequent analysis of serum copper content.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all live pups per litter
- Soft tissue examinations: Yes: all live pups per litter
- Skeletal examinations: Yes: half of live pups per litter
- Head examinations: Yes: half of live pups per litter
Statistics:
The unit of comparison was the pregnant female or the litter (Weil, 1970). Results of the quantitative continuous variables (e.g., maternal body weights, organ weights, fetal weights, etc.) were intercompared for the three triethylenetetramine-exposed groups and the vehicle control group by use of Levene's test for equal variances (Levene, 1960), analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances and the ANOVA was significant, the pooled t-test was used, When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Brown and Forsythe, 1974) followed, when necessary, by the separate variance t-test.
Nonparametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test (Sokal and Rohlf, 1969) followed by the Mann-Whitney U test (Sokal and Rohlf, 1969) when appropriate, Incidence data were compared using Fisher's Exact Test (Sokal and Rohlf, 1969). For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.
Indices:
pregnancy rate,
number of totally resorbed litters,
number of ovarian corpora lutea of pregnancy,
total implantations,
viable or non-viable implantations per litter,
percent preimplantation loss,
percent live fetuses per litter,
sex ratio ( % males).
Historical control data:
No data given in the report
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No effect on gestational parameters. There were no differences among groups for either maternal serum or urine copper.

Maternal body weight was significantly reduced at 125.0 mg/kg/day for gd 18 (last treatment day). Maternal weight gain was significantly reduced only at 125.0 mg/kg/day for gd 12-15, 15-18 (latter half of the treatment period), and gd 6-18 (treatment period). The body weight gain during the post exposure period (gd 18-29) indicated that the high level animals gained more weight than the vehicle controls, but this value was not statistically significant. By sacrifice on gd 29, there were no statistically significant differences among groups for body weight.

Statistically significant increases in incidence were observed for full feed hoppers in the post-treatment period (indirect evidence for reduced food consumption) at 125.0 mg/kg/day (two of the five does with this finding subsequently died) and ecchymosis (subepidermal hematomas), necrosis and fissures at the dosing site during and after the treatment period at 50.0 and 125.0 mg/kg/day. The two does at 125.0 mg/kg/day which died also exhibited red urogenital discharge, emaciation (the doe who died on gd 20) and blood on paperboard (the doe who died on gd 29).

A dose-related pattern of erythema and edema. The high level (125.0 mg/kg/day) does were the first to show signs of skin irritation (gd 6) and after the treatment stopped, they were the last to show signs of healing. The low (5.0 mg/kg/day) and middle (50.0 mg/kg/day) dose groups showed lesser signs of irritation on gd 7 (does at 50.0 mg/kg/day exhibited only minimal signs of erythema and no edema on gd 6). The edema present during dosing on the does from the 5.0 mg/kg/day dose group ceased after treatment was stopped (resolved by gd 21) and the erythema healed almost completely by gd 23. The does at the middle and high dose levels never completely healed from the edema and erythema caused by treatment. The vehicle control group exhibited slight erythema and edema from the procedures (never more than a grade of 1 for both parameters).

At scheduled sacrifice on gd 29, the only statistically significant gross observations due to treatment were increases in the incidence of necrosis and ecchymosis at 50.0 and 125.0 mg/kg/day at the dosing site. Also at sacrifice, terminal body weight, corrected terminal body weight, and gestational weight change (both of the latter parameters corrected for the weight of the gravid uterus), gravid uterine weight and absolute and relative liver, lung and kidney weights were unaffected by treatment.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality
Dose descriptor:
LOAEL
Remarks:
local
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
dermal irritation
other: minimal skin effects which had completely recovered at the end of the study
Abnormalities:
effects observed, treatment-related
Localisation:
other: dermal irritation
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean litter weight, male or female fetal body weights per litter, taken at sacrifice on gd 29, were unaffected by treatment.

There was no significant increase in the number of litters with one or more affected fetuses in any exposure group relative to the vehicle control group for individual and total external, visceral (including craniofacial) and skeletal malformations, or total malformations (all categories combined). There were no treatment-related changes in the incidence of any external or visceral variations reported by individual findings or by category.

The incidence of one skeletal variation in triethylenetetramine-exposed groups differed significantly from that in the vehicle control group out of 73 findings. That finding was a decreased incidence in body of the hyoid bone poorly ossified. The significance of this finding is unknown but is probably due to biological variation. No other skeletal variations exhibited significant differences in incidence among groups.
Dose descriptor:
NOAEL
Effect level:
>= 125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Administration of triethylenetetramine by occluded cutaneous application to timed-pregnant New Zealand white rabbits during organogenesis resulted in maternal toxicity at 125.0 mg/kg/day, i.g. mortality (9.1%), transient reduced weight gain during exposure and clinical signs of toxicity. Only lesions at the dosing site were observed at 50.0 mg/kg/day. There was no maternal toxicity at 5.0 mg/kg/day except for transient irritation at the application site. There were no effects of treatment on maternal serum or urinary copper content assayed at sacrifice. There was no evidence of developmental toxicity (including teratogenicity) at any dose level employed.
Executive summary:

Timed-pregnant New Zealand White rabbits were treated with triethylenetetramine by occluded cutaneous application on gestational days (gd) 6 through 18, six hours per day. Groups of 22 does each received triethylenetetramine formulated in deionized water and applied in a volume of 2.0 ml/animal (approximately 0.5 ml/kg) at dosages of 0.0, 5.0, 50.0 or 125.0 mg/kg/day. Clinical observations of all study animals were undertaken daily and maternal body weights were measured on gd 0, 6, 12, 15, 18 and 29. In addition, daily observations of the dosing site of treated animals were recorded from the initiation of the treatment period and continued until the termination of the study. At scheduled necropsy on gd 29, does were evaluated for body weight, liver weight, lung and kidney weight (2), gravid uterine weight and status of implantation sites (i.e., resorptions, dead fetuses, live fetuses). Maternal blood and urine were evaluated for copper content. Live fetuses were dissected from the uterus, counted, weighed, examined for external, visceral and skeletal abnormalities and sexed internally. Maternal kidneys and liver sections were retained in fixative for possible subsequent histopathologic examination. Two does (9.1%) died at 125.0 mg/kg/day. Maternal toxicity was also indicated by a significant decrease in body weight gain (in the exposure period) at 125.0 mg/kg/day. Also, clinical observations indicated severe skin irritation at 50.0 and 125.0 mg/kg/day with some recovery in the post-dosing period, Slight irritation at the dosing site was observed at 5.0 mg/kg/day which was transient, with essentially total recovery after exposures ceased. There were no effects on maternal lung, kidney or liver weights (absolute or relative to body weight) or on gravid uterine weight. There were no differences among groups for maternal serum or urinary copper. There was no effect of treatment on number of ovarian corpora lutea, number of total, live, or non-viable implantations per litter, or percent preimplantation loss, percent live fetuses or sex ratio (% male fetuses). Fetal body weights per litter (males, females or total) were also unchanged across groups, There was no significant increase in the incidence of individual malformations or variations or of external, visceral, skeletal, or total (external, visceral, plus skeletal) malformations or variations by fetus or by litter in any triethylenetetramine exposed group relative to the vehicle control group. One finding, poorly ossified hyoid bone exhibited a decreased incidence at 125.0 mg/kg/day relative to that in the vehicle control group. Thus, under the conditions of this study, triethylenetetramine produced maternal toxicity other than at the dosing site only at 125.0 mg/kg/day but no developmental toxicity at any dosages employed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is comparable to guideline, with Klimisch score 2.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The key study is GLP compliant and comparable to guideline, with Klimisch score 2.
Additional information

For Amines, polyethylenepoly-, triethylenetetramine fraction two reliable studies according to OECD guideline 414 in rats and rabbits are available.

Timed-pregnant Sprague Dawley rats were treated with Amines, polyethylenepoly-, triethylenetetramine fraction by daily oral gavage on gestational days (gd) 6 up to and including day 15 (CIBA-GEIGY, 1984). Groups of 24 females each received the test substance formulated in distilled water applied at a volume of 10 mL/kg bw at dosages of 0, 75, 325 or 750 mg/kg bw per day. With regard to the vehicle control, food intake was slightly depressed in all dose groups during the second part of the treatment period. Body-weight gain appeared to be somewhat reduced in the experimental groups; however when corrected body weights were evaluated no differences were observed. No other toxic signs were observed in the mothers. A significant increase in the average body weight of the live foetuses was recorded for the high-dose group in the main study. The gross inspection of the live foetuses did not reveal any finding either in the experimental groups or in the vehicle control. The skeletal assessment revealed instances of irregular sternebral ossification in the dose groups and in the vehicle control. Concerning the status of skeletal maturation of the foetuses shortly before term, apart from a slightly raised number of still unossified calcanei in the high-dose group in the main study, no delay of ossification could be found for the experimental groups in comparison with the vehicle control.

Thus, under the conditions of this study, the test substance produced no maternal toxicity except for slightly reduced food intake and no developmental toxicity at any dosages employed. Therefore, the NOAEL for maternal toxicity and developmental toxicity was considered to be equal or greater than 750 mg/kg bw/day in rats after oral exposure.

Timed-pregnant New Zealand White rabbits were treated with Amines, polyethylenepoly-, triethylenetetramine fraction by occluded cutaneous application on gestational days (gd) 6 through 18, six hours per day (Tyl, 1988). Groups of 22 does each received the test substance formulated in deionized water and applied in a volume of 2.0 mL/animal (approximately 0.5 mL/kg bw) at dosages of 0.0, 5.0, 50.0 or 125.0 mg/kg bw/day. Clinical observations of all study animals were undertaken daily and maternal body weights were measured on gd 0, 6, 12, 15, 18 and 29. In addition, daily observations of the dosing site of treated animals were recorded from the initiation of the treatment period and continued until the termination of the study. At scheduled necropsy on gd 29, does were evaluated for body weight, liver weight, lung and kidney weight, gravid uterine weight and status of implantation sites (i.e., resorptions, dead fetuses, live fetuses). Maternal blood and urine were evaluated for copper content. Live fetuses were dissected from the uterus, counted, weighed, examined for external, visceral and skeletal abnormalities and sexed internally. Maternal kidneys and liver sections were retained in fixative for possible subsequent histopathologic examination. Two does (9.1%) died at 125.0 mg/kg bw/day. Maternal toxicity was also indicated by a significant decrease in body weight gain (in the exposure period) at 125.0 mg/kg bw/day. Also, clinical observations indicated severe skin irritation at 50.0 and 125.0 mg/kg bw/day with some recovery in the post-dosing period. Slight irritation at the dosing site was observed at 5.0 mg/kg bw/day which was transient, with essentially total recovery after exposures ceased. There were no effects on maternal lung, kidney or liver weights (absolute or relative to body weight) or on gravid uterine weight. There were no differences among groups for maternal serum or urinary copper. There was no effect of treatment on number of ovarian corpora lutea, number of total, live, or non-viable implantations per litter, or percent preimplantation loss, percent live fetuses or sex ratio (% male fetuses). Fetal body weights per litter (males, females or total) were also unchanged across groups. There was no significant increase in the incidence of individual malformations or variations or of external, visceral, skeletal, or total (external, visceral, plus skeletal) malformations or variations by fetus or by litter in any triethylenetetramine exposed group relative to the vehicle control group. One finding, poorly ossified hyoid bone exhibited a decreased incidence at 125.0 mg/kg bw/day relative to that in the vehicle control group. Thus, under the conditions of this study, the test substance produced maternal toxicity other than at the dosing site only at 125.0 mg/kg bw/day but no developmental toxicity at any dosages employed.

Thus, under the conditions of this study, the test substance resulted in mortality in the highest dose group. No developmental toxicity at any dosages employed was observed. Therefore, the NOAEL for maternal toxicity and was considered to be 50 mg/kg bw/day and the NOAEL for developmental toxicity equal or greater than 125 mg/kg bw/day in rabbits after dermal exposure.

Four further publications are available where the potential for developmental toxicity of Amines, polyethylenepoly-, triethylenetetramine fraction was examined. All those data are disregarded as they show significant methodological deficiencies. One evaluation was performed in chicken embryos (Korhonen, 1983). The test substance was injected in chicken embryo eggs once. Injections of the test substance resulted in death and malformation of the embryos indicating a developmental toxicity effect. This is not a validated test method thus no conclusion on the developmental toxicity can be drawn. As no developmental effects were observed in rabbits and rats this result is considered to be not relevant. Furthermore, three publications are available where the developmental toxicity of Amines, polyethylenepoly-, triethylenetetramine fraction is examined in guinea pigs and rats after dermal exposure during pregnancy (Szacki, 1974, Dobryszycka, 1975, 1974). No detailed examination of maternal animals and fetuses were performed. Only one dose was used which revealed severe skin reactions. Due to those methodological deficiencies no conclusion on developmental toxicity effects of Amines, polyethylenepoly-, triethylenetetramine fraction can be drawn.

In summary, data from studies with Amines, polyethylenepoly-, triethylenetetramine fraction do not provide evidence of developmental toxicity.


Justification for classification or non-classification

The available data on developmental toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification. However, no final decision on classification for toxicity to reproduction according to Regulation (EC) 1272/2008 can be made, as no information on fertility is available.