Amines, (2-ethylhexyl)(hydrogenated tallow alkyl)methyl

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 8, 2016 to May 31, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test
system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for
reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive
toxicants.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of
treatment for the males and females. A breeding record containing the male and female
identification numbers and the start date of cohabitation was maintained. Each female was
cohabitated with 1 male in a solid-bottom cage containing bedding material. Positive evidence
of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm
following a vaginal lavage and verified by a second biologist. Each mating pair was examined
daily. The day when evidence of mating was identified was termed Gestation Day 0. If
evidence of copulation was not detected after 14 days of pairing, any females that had not shown
evidence of mating were placed in solid-bottom cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item homogeneity and stability following at least 10 days of room temperature storage at
concentrations of 20 to 200 mg/mL were previously established. An extension validation of test
item homogeneity and stability at a concentration of 2.00 mg/mL was conducted in a concurrent
study to cover the lowest concentration used in the current study.
Prior to the initiation of dose administration, samples for homogeneity and/or concentration
determination were collected from the top, middle, and bottom strata of the first 2 and
200 mg/mL formulations and from the middle stratum of the control, 8, and 40 mg/mL
formulations. In addition, samples for resuspension homogeneity and stability determinations
were collected from the top and bottom strata of an aliquot taken from the 2 and 200 mg/mL
suspensions following room temperature storage for 5 and 11 days; aliquots were mixed for a minimum of 30 minutes prior to sample
collection. Samples for concentration analyses were also collected from the middle stratum of
the middle and last formulations (including the control group) prepared during the study. One
set of samples from each collection was subjected to the appropriate analyses. All remaining
samples were stored at room temperature as back-up. All analyses were conducted by the
Charles River Analytical Chemistry Department using a validated high performance liquid
chromatography method using charged aerosol detection.

Duration of treatment / exposure:

The males were dosed during Study Days 0 through 27 or 28 (14 days prior to pairing
through 1 day prior to scheduled euthanasia) for a total of 28–29 doses. The females were dosed
from Study Day 0 through the day prior to euthanasia (14 days prior to pairing through Lactation
Day 13) for a total of 49–53 doses. Females that failed to deliver were dosed through the day
prior to euthanasia (Post-Mating Day 25) for a total of 39–41 doses.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Parental animals: Observations and examinations:

Clinical observation and survival - twice daily and 1-2 hours post-dosing
Body weights - weekly and more frequently for females during gestation
Food consumption - weekly

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to
determine the stage of the estrous cycle of each female for 14 days prior to test item
administration and continuing until evidence of copulation was observed. The average cycle length was calculated and reported for
complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from
estrus [E] or proestrus [P] until the detection of evidence of mating), beginning with the first day
of dose administration. Estrous cycle length was determined by counting the number of days
from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during
which evidence of mating was observed for a given animal was not included in the mean
individual estrous cycle length calculation.

Sperm parameters (parental animals):

Not evaluated

Litter observations:

Litter viability, size, and death
Clinical observations
Body weights
Sex determination
Litter parameters (live liter size, postnatal survival)
Thyroid hormone analysis

Postmortem examinations (parental animals):

Clinical pathology (hematology, coagulation, serum chemistry) collected from 5 animals/sex/group at scheduled necropsies.
Complete necropsy condcuted on all parental animals
Thyroid hormone analysis

Postmortem examinations (offspring):

On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium
pentobarbital. Blood samples were collected for thyroid hormone analysis immediately prior to
euthanasia from 1 pup/sex/litter; the thyroids (with parathyroids, if present)
were weighed (following fixation) and placed in 10% neutral-buffered formalin for possible
histopathological examination. Remaining pups (not used for blood collection) were discarded without examination.

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

3 females in the 1000 mg/kg/day were euthanized in extremis

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related, significantly (p < 0.05) higher mean absolute neutrophil counts were noted in
the 1000 mg/kg/day group F0 males and correlated with histopathologic findings in the lungs and
nasal cavity.
Test item-related, significantly (p < 0.05 or p < 0.01) higher mean absolute neutrophil counts
were also noted in the 10, 40, and 200 mg/kg/day group F0 females. There were no
histopathologic correlates and, thus, the cause of the increase in neutrophil counts was uncertain
in the females.
There were no other test item-related effects on hematology or coagulation parameters.
However, some statistically significant differences were observed when the control and test
item-treated groups were compared.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related, significantly (p < 0.05 or p < 0.01) lower mean serum albumin values were
noted in the 200 and 1000 mg/kg/day group F0 males. Test item-related, significantly (p < 0.05
or p < 0.01) lower mean serum protein values were also noted in the 200 and 1000 mg/kg/day
group F0 males and were considered secondary to the lower mean albumin values.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the non-glandular stomach, lungs, and nasal
cavity of the 1000 mg/kg/day group F0 males and females and in the glandular stomach, thymus,
spleen, Peyer’s Patches, mesenteric lymph node, bone marrow (sternum), and adrenal cortex of
the 1000 mg/kg/day group F0 females.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A complete evaluation of maternal reproductive toxicity was precluded by the fact that none of
the females at 1000 mg/kg/day survived to the scheduled necropsy on Lactation Day 14.
Interpretation of the limited amount of reproductive data available for the 1000 mg/kg/day group
females was likely confounded by the presence of maternal toxicity. F0 male and female mating
indices in the 1000 mg/kg/day group were similar to the control group. Test item-related lower
mean F0 male and female fertility, male copulation, and female conception indices were noted in
the 1000 mg/kg/day group compared to the control group; differences for the fertility and male
copulation indices were significant (p < 0.05) and all values in the 1000 mg/kg/day group were
below the minimum mean values in the Charles River Ashland historical control data. In the
1000 mg/kg/day group, 5 males did not sire a litter and 5 out of the 9 females that mated were
determined to be nongravid. There were 2 gravid females that failed to deliver due to having
totally resorbed litters (Nos. 6981 and 7017) and 1 gravid female that was euthanized in extremis
during gestation (No. 7040) in the 1000 mg/kg/day group.
No test item-related effects on reproductive performance were observed in the 10, 40, and
200 mg/kg/day groups. Differences from the control group were slight and not statistically
significant. All males in the 10, 40, and 200 mg/kg/day groups sired a litter and all females in
the same respective groups were determined to be gravid.
The mean numbers of days between pairing and coitus in the test item-treated groups were
similar to the control group value. The mean lengths of estrous cycles in these groups were also
similar to the control group value. None of these differences were statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

other: day based on no adverse effects at this dose level and an inability to fully evaluate the 1000 mg/kg/day group due to severe maternal toxicity and failure to deliver (including resorbed litters, being nongravid, or total litter loss).

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

other: adverse microscopic findings (hyperplasia, ulceration, necrosis, and neutrophil inflammation in the glandular and non-glandular stomach and decreased lymphoid cellularity in the thymus and spleen)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of systemic toxicity (highest dose tested)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical findings) of all
F1 pups in this study was unaffected by parental administration of the test item. Pups (litters)
that were found dead numbered 4(3), 7(2), 4(4), 3(1), and 15(1) in the control, 10, 40, 200, and
1000 mg/kg/day groups, respectively. One, 2, 1, and 12 pups in the control, 10, 40, and
200 mg/kg/day groups, respectively, was missing and presumed to have been cannibalized.

Mortality / viability:

no mortality observed

Description (incidence and severity):

In the 1000 mg/kg/day group, all females were euthanized in extremis during the breeding period
or during gestation, failed to deliver (due to totally resorbed litters or being nongravid), or had a
total litter loss, and therefore no viable pups were available for evaluation of PND 0 litter data
and postnatal survival in this group.
A significantly (p < 0.05) lower mean number of pups born was noted in the 200 mg/kg/day
group compared to the concurrent control group. However, this difference was attributed to a
slightly (not significantly) lower mean number of implantation sites in this group, and not to
postimplantation loss. The mean number of pups born at 200 mg/kg/day (12.3 pups/litter), while
slightly lower than the concurrent control group, was within the Charles River Ashland historical
control data range (11.8–16.6 pups/litter). The mean number of pups born in the concurrent
control group (15.3 pups/litter) was also greater than 1 standard deviation of the historical
control mean (14.2 ± 0.85). Mean live litter size (PND 0) was similar to the concurrent control
group at 200 mg/kg/day; the difference was not statistically significant.
The mean number of pups born and live litter size in the 10 and 40 mg/kg/day groups and the
percentage of males at birth in the 10, 40, and 200 mg/kg/day groups were generally similar to
the control group values. Postnatal survival in the 10, 40, and 200 mg/kg/day groups were
unaffected by test item administration. Lower (not statistically significant) mean postnatal
survival was noted in the 200 mg/kg/day group compared to the control group during PND 0–1
and from birth to PND 4 (pre-selection). However, these differences were primarily attributed to
a single female in this group (No. 7033) that had a total litter loss on Lactation Day 1; therefore,
the differences noted in the 200 mg/kg/day group were not considered test item-related.

Body weight and weight changes:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no viable pups in the 1000 mg/kg/day group; therefore, evaluation of organ weight
data at the scheduled pup necropsy on PND 13 was precluded for this group.
On PND 13, test item-related lower mean thyroid/parathyroid weights were noted in the
200 mg/kg/day group F1 males and females compared to the control group; differences were
significant (p < 0.05 or p < 0.01). In the absence of any other signs of neonatal toxicity, these
relatively small differences in thyroid/parathyroid weights in the 200 mg/kg/day group were not
considered to be adverse.
There were no test item-related effects on thyroid/parathyroid weights in the F1 males and
females in the 10 and 40 mg/kg/day groups on PND 13. Differences in the 10 and 40 mg/kg/day
groups from the control group were considered to be the result of normal biological variation and
were not considered to be of toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No internal findings that could be attributed to parental test item administration were noted at the
necropsy of pups euthanized on PND 13. The only internal finding was dark red discoloration of
the mandibular salivary gland noted for Male Pup No. 6996-02 and Female Pup No. 6996-06 in
the 200 mg/kg/day group; because this finding was noted infrequently in a single litter, it was not
considered test item-related.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no test item-related effects on thyroid hormone values in the F1 males and females at
any dosage level for culled pups on PND 4 or on PND 13. Differences from the control group
were considered to be the result of normal biological variation and were not considered to be of
toxicological significance.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on no adverse effects at this dose level an no evaluation of the F1 litter data due to early termination of the 1000 mg/kg/day dose group resulting in no viable pups.

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

no

Relevant for humans:

no

Test item-related effects occurred on the physical condition and survival of F0 females in the

1000 mg/kg/day group. Three females in the 1000 mg/kg/day group were euthanized in extremis

during the breeding period (Study Day 21) or during gestation (Gestation Days 10 and 21)

following adverse clinical signs (thin, pale, and/or cool body) and/or severe body weight losses

prior to euthanasia. At necropsy, a cause of death could not be determined for these females, but

associated test item-related microscopic findings included the following, some of which are

considered to be adverse: hyperplasia in nonglandular stomach, nasal degeneration, neutrophil

inflammation in the nasal cavity, decreased lymphoid cellularity in the thymus and/or

mandibular lymph node, decreased cellularity in the bone marrow; vacuolation and hypertrophy

of the zona fasciculate in adrenal gland cortex; and mixed cell inflammation and type II

pneumocyte hyperplasia in the lungs. One female in the 1000 mg/kg/day group was euthanized

early soon after parturition due to a total litter loss on Postnatal Day (PND) 0. This female was

noted with pale body and cool extremities and microscopic findings, including ulceration,

necrosis and neutrophil inflammation in glandular and non-glandular stomach and intestine;

presence of bacterial and fungal organisms, indicating that the animal was immunosuppressed,

and findings in lymph node, bone marrow, and adrenal cortex attributed to stress response. The

6 remaining females in this group failed to deliver and were necropsied on Post-Mating Day 25;

2 were found to have been gravid. The microscopic findings for these females are summarized

in the text table below. Because the moribundity and unscheduled deaths noted at

1000 mg/kg/day occurred only in females at the highest dosage level and were associated with

clinical and microscopic findings, they were considered test item-related. A complete evaluation

of maternal systemic and reproductive toxicity was precluded by the fact that none of the females

at 1000 mg/kg/day survived to the scheduled necropsy on Lactation Day 14. Interpretation of the

limited amount of reproductive data available for the 1000 mg/kg/day group females was also

confounded by the presence of maternal toxicity at this same dosage level.

All F0 females in the control, 10, 40, and 200 mg/kg/day groups and all F0 males in the control,

10, 40, 200, and 1000 mg/kg/day groups survived to the scheduled necropsies; no test

item-related clinical observations were observed in these same groups at the daily examinations,

weekly detailed examinations, or approximately 1–2 hours following dose administration at any

dosage level.

Mean body weights, body weight gains, and food consumption for F0 females were unaffected by

test item administration during the pre-mating period (Study Days 0–13). In the 200 and

1000 mg/kg/day group F0 females, mean body weight gains were similar to the control group

during Gestation Days 0-7 followed by lower mean body weight gains during the remainder of

gestation (Gestation Days 7-11 and 17-20 at 200 mg/kg/day and Gestation Days 7-20 at

1000 mg/kg/day). The decrements in mean body weight gain during the latter half of gestation

resulted in lower mean body weight gains at 200 and 1000 mg/kg/day when the entire gestation

treatment period (Gestation Days 0–20) was evaluated compared to the control group. In

addition, mean body weights in the 200 and 1000 mg/kg/day groups were 6.9% and 17.3%

lower, respectively, than the control group on Gestation Day 20. The aforementioned body

weight differences at 1000 mg/kg/day correlated with 3 females being euthanized in extremis

during breeding or gestation, the failure of 6 females to deliver (due to totally resorbed litters or

being nongravid), and a total litter loss for 1 female on the same day parturition was complete.

The test item-related effects on mean female body weight at 200 mg/kg/day during gestation

were transient and of small magnitude (≤ 6.9%; not statistically significant), and therefore not

considered adverse. Mean food consumption in the 1000 mg/kg/day group was slightly lower

than the control group during Gestation Days 14-20; mean food consumption was similar to the

control group during Gestation Days 0–14 at 1000 mg/kg/day and throughout gestation at

200 mg/kg/day. All females in the 1000 mg/kg/day group were euthanized in extremis during

the breeding period or gestation, failed to deliver either due to resorbed litters or being

nongravid, or had a total litter loss; therefore, evaluation of lactation body weight and food

consumption parameters was precluded in this group. Lower mean body weights and body

weight gains with reduced food consumption were noted in the 200 mg/kg/day group at the

beginning of lactation (Lactation Days 1-4); mean body weight and food consumption

parameters in this group were similar to the control group for the remainder of the lactation

treatment period (Lactation Days 4-13). The effect on mean female food consumption at

200 mg/kg/day during lactation was transient and not of sufficient magnitude to adversely impact

lactational body weight; therefore, these differences at 200 mg/kg/day were not considered to be

adverse. There were no test item-related effects on mean body weights, body weight gains, and

food consumption for F0 males at any dosage level throughout the study or in the 10 and 40

mg/kg/day group F0 females during gestation and lactation.

In the 1000 mg/kg/day group, there were no F0 females available for the FOB or motor activity

assessments on Lactation Day 13; therefore, evaluation of these parameters was precluded for

females in this group. No test item-related effects were noted during the FOB or motor activity

evaluations at any dosage level for males and females evaluated in the F0 generation.

Test item-related lower mean F0 male and female fertility (44.4%), male copulation (44.4%), and

female conception indices (44.4%) were noted in the 1000 mg/kg/day group due to 5 out of

9 mated females being nongravid at necropsy; male and female mating indices in this group were

100.0%. In the 1000 mg/kg/day group, 5 males did not sire a litter and 5 females were

determined to be nongravid. In addition, there were 2 gravid females that failed to deliver (due

to having totally resorbed litters) in this group. Two females (Nos. 7040 and 7038; see text table

below) in the 1000 mg/kg/day group were noted with pale and cool body and/or extremities

during parturition on Gestation Day 21 or the same day parturition was completed (PND 0); no

other signs of dystocia were noted at any dosage level. F0 estrous cycle length and the mean

number of days between pairing and coitus at all dosage levels and reproductive performance

and gestation lengths at 10, 40, and 200 mg/kg/day were unaffected by test item administration.

There were no test item-related effects on the mean number of implantation sites and

unaccounted-for sites at 10, 40, and 200 mg/kg/day.

There were no viable pups in the 1000 mg/kg/day group; therefore, evaluation of F1 offspring

data was precluded in this group. The mean number of F1 pups born in the 200 mg/kg/day group

was significantly lower than the concurrent control group; however, the mean number of pups

born at this dosage level was within the range of values in the Charles River Ashland historical

control data, and there were no statistically significant differences relative to the concurrent

control group for mean numbers of implantation sites, unaccounted sites, or live litter size on

PND 0 at 200 mg/kg/day. One female (No. 6988) in the 200 mg/kg/day group also had only

7 live pups. There were no test item-related effects on the number of F1 pups born and live litter

size at 10 and 40 mg/kg/day.

The percentage of males at birth, F1 clinical observations, postnatal survival and growth,

anogenital distance, and areolae/nipple anlagen were unaffected at 10, 40, and 200 mg/kg/day.

There were also no test item-related macroscopic findings for F1 pups that were found dead or at

the PND 13 scheduled necropsy at any dosage level.

Lower mean thyroid/parathyroid weights were noted in the 200 mg/kg/day group F1 male and

female pups on PND 13 and were considered test item-related, but in the absence of any other

signs of neonatal toxicity, these relatively small differences in thyroid/parathyroid weights were

not considered to be adverse. There were no test item-related changes in thyroid/parathyroid

weights in the 10 and 40 mg/kg/day groups.

Test item-related histopathological findings of squamous cell hyperplasia and hyperkeratosis

(1000 mg/kg/day F0 males only) in the non-glandular stomach were noted in F0 males at 200 and

1000 mg/kg/day and in F0 females at 1000 mg/kg/day. Epithelial degeneration accompanied by

neutrophil inflammation was also noted in the non-glandular stomach of F0 males at

200 mg/kg/day, while mixed inflammation with type II pneumocyte hyperplasia in the lungs and

epithelial degeneration with neutrophil inflammation in the nasal cavity were noted in F0 males

and females at 1000 mg/kg/day. The aforementioned findings were considered due to local

irritation and correlated with increased neutrophil counts in the 1000 mg/kg/day group F0 males.

Test item-related macrophage hyperplasia in the medulla of the mesenteric lymph node was also

noted in the 1000 mg/kg/day group F0 males and females. All of the aforementioned findings

were not considered adverse.

Indirect, likely stress-related, test item-related findings of decreased lymphoid cellularity

(thymus, spleen, Peyer’s patches, mesenteric lymph node); decreased bone marrow cellularity;

increased vacuolation and hyperplasia of the adrenal gland; and ulceration, necrosis, and

neutrophil inflammation of the glandular and non-glandular stomach were noted in 1 F0 female at

1000 mg/kg/day that had a total litter loss. The findings in the glandular and non-glandular

stomach, thymus, and spleen were considered adverse, while findings in the Peyer’s patches,

mesenteric lymph node, bone marrow, and adrenal cortex were not considered adverse.

Additional test item-related clinical pathology findings in the F0 generation included lower mean

serum albumin and total protein values in the 200 and 1000 mg/kg/day group males, lower mean

T4 values in the 40, 200, and 1000 mg/kg/day group males, and higher mean neutrophil counts in

the 10, 40, and 200 mg/kg/day group females. Test item-related lower mean heart weights were

noted in the 200 mg/kg/day group females. Alterations in the serum albumin and total protein

values and heart weights were not considered adverse. The adversity regarding the alterations in

T4 values and neutrophil counts could not be ascertained without additional information;

however, there were no correlating histopathologic findings or alterations in organ weights.

Conclusions:

While no females in the 1000 mg/kg/day group successfully delivered pups, this occurred in the presence of severe maternal toxicity that, according to GHS/CLP guidance, should not be considered in the hazard classification. Following is an excerpt from the GHS guidance on maternal mortality:

Maternal mortality: an increased incidence of mortality among the treated dams over the controls shall be considered evidence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the systemic toxicity of the test material. Maternal mortality greater than 10 % is considered excessive and the data for that dose level shall not normally be considered for further evaluation.

3/10 animals in the 1000 mg/kg/day dose group were euthanized in extremis during the study period (> 10% GHS/CLP criteria) and one additional animal had severe toxicity/stress; of the remaining 6 animals, 4 were non-gravid leading to a low fertility index (44.4%), which is another indicator of maternal toxicity per GHS/CLP guidance.

No other evidence or signs of reproductive or developmental toxicity were observed or noted in the OECD 422 study with the notified substance. In addition, no signs of reproductive toxicity (e.g., reproductive organ weight) was observed in the OECD 408 90-day repeat dose study with the notified substance. As such, it is unlikely that the notified substance is causing specific or targeted reproductive or developmental toxicity.

Executive summary:

Evaluation of maternal systemic and reproductive toxicity was precluded in this study because

none of the F0 females at 1000 mg/kg/day survived to scheduled necropsy on Lactation Day 14.

All females in the 1000 mg/kg/day group were euthanized in extremis during the breeding period

prior to mating or during gestation, failed to deliver either due to resorbed litters or being

nongravid, or had total litter loss. As a result, the reproductive data available for interpretation

were limited for the 1000 mg/kg/day group and were likely confounded by the presence of

maternal systemic toxicity, including adverse clinical findings, severe body weight deficits

during gestation, and adverse microscopic findings (hyperplasia, ulceration, necrosis, and

neutrophil inflammation in the glandular and non-glandular stomach and decreased lymphoid

cellularity in the thymus and spleen). Based on these results, the no-observed-adverse-effect

level (NOAEL) for F0 female systemic toxicity was considered to be 200 mg/kg/day. While test

item-related effects on F0 female body weight and food consumption were noted at

200 mg/kg/day, the differences were small, transient, and not considered to be adverse. Based

on the absence of systemic toxicity for the F0 males at any dosage level, the NOAEL for male

systemic toxicity was considered to be 1000 mg/kg/day, the highest dosage level tested. While

histopathological findings in the non-glandular stomach were noted in F0 males at 200 and

1000 mg/kg/day, these findings were attributed to local irritation of the test item and not

considered adverse. Clinical pathology findings in the F0 generation included lower mean serum

albumin and total protein values in the 200 and 1000 mg/kg/day group males, lower mean T4

values in the 40, 200, and 1000 mg/kg/day group males, and higher mean neutrophil counts in

the 10, 40, and 200 mg/kg/day group females. Lower mean heart weights were also noted in the

200 mg/kg/day group females. Alterations in the serum albumin and total protein values and

heart weights were not considered adverse. The adversity regarding the alterations in T4 values

and neutrophil counts could not be ascertained without additional information; however, there

were no correlating histopathologic findings or alterations in organ weights.

Because a complete evaluation of F0 reproductive toxicity was precluded at 1000 mg/kg/day by

maternal toxicity and early termination of all F0 females prior to scheduled necropsy, a dosage

level of 200 mg/kg/day was considered to be the NOAEL for male and female reproductive

toxicity of Amines (2-ethylhexyl)(hydrogenated tallow alkyl) methyl when administered orally

by gavage to Crl:CD(SD) rats. Test item-related lower fertility indices were noted in the

1000 mg/kg/day group due to 5 out of 9 mated females that were nongravid. Two gravid females

in the 1000 mg/kg/day group had totally resorbed litters and one other female had a total litter

loss on the day parturition was complete. These reproductive findings occurred at a dosage level

that also produced adverse maternal toxicity. F0 mating indices (100%), estrous cycle length,

and the number of days between pairing and coitus at 1000 mg/kg/day were unaffected by test

item administration.

Because of the early termination of all F0 females, no viable pups were available for evaluation

of F1 litter data at 1000 mg/kg/day. Therefore, the NOAEL for neonatal toxicity was considered

to be 200 mg/kg/day. The mean number of F1 pups born at 200 mg/kg/day was significantly

lower than the concurrent control group; however, this value was within the range of values in

the Charles River Ashland historical control data and there were no statistically significant

effects on numbers of implantation sites, unaccounted sites, or live litter size on PND 0 at

≤ 200 mg/kg/day. The percentage of males at birth, F1 clinical observations, postnatal survival

and growth, anogenital distance, and areolae/nipple anlagen were unaffected at dosages

≤ 200 mg/kg/day. Lower mean thyroid/parathyroid weights were noted in the 200 mg/kg/day

group F1 male and female pups on PND 13, but in the absence of any other signs of neonatal

toxicity, these relatively small differences in thyroid/parathyroid weights were not considered

adverse.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

While no females in the 1000 mg/kg/day group successfully delivered pups, this occurred in the presence of severe maternal toxicity that, according to GHS/CLP guidance, should not be considered in the hazard classification. Following is an excerpt from the GHS guidance on maternal mortality:

Maternal mortality: an increased incidence of mortality among the treated dams over the controls shall be considered evidence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the systemic toxicity of the test material. Maternal mortality greater than 10 % is considered excessive and the data for that dose level shall not normally be considered for further evaluation.

3/10 animals in the 1000 mg/kg/day dose group were euthanized in extremis during the study period (> 10% GHS/CLP criteria) and one additional animal had severe toxicity/stress; of the remaining 6 animals, 4 were non-gravid leading to a low fertility index (44.4%), which is another indicator of maternal toxicity per GHS/CLP guidance.

No other evidence or signs of reproductive or developmental toxicity were observed or noted in the OECD 422 study with the notified substance. In addition, no signs of reproductive toxicity (e.g., reproductive organ weight) was observed in the OECD 408 90-day repeat dose study with the notified substance. As such, it is unlikely that the notified substance is causing specific or targeted reproductive or developmental toxicity.