Reaction mass of Copper,di-µ-iodotris[triphenylphosphine]di- and iodobis(triphenylphosphino)copper

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the results obtained in this study the NOAEL (No Observed Adverse Effect Level) for

both general toxicity and reproduction/developmental toxicity was considered to be 1000

mg/kg/day for males and females.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/2017 to 03/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The dose levels of 100, 300 and 1000 mg/kg/day were selected in consultation with the
Sponsor based on information from a preliminary non-GLP compliant study (RTC Study no.
E0107). In this study, the test item was administered for 2 weeks at dose levels of 100, 300
and 1000 mg/kg bw/day in male and female rats. No clinical signs, no effects on body weight
and food consumption, no changes in organ weight and no treatment-related macroscopic
observation were noted in the treated animals, when compared to controls.

Specific details on test material used for the study:

Identity: Iodobis(triphenylphosphino)copper
Batch no.: 16101213
CAS no.: 16109-82-3
EC no.: 240-276-1
Expiry date: 12 December 2017
Storage conditions: Room temperature
RTC number: 15248
Trade name: Bruggolen H3301

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

The Sprague Dawley SD rat was the species and strain of choice because it is accepted by
many regulatory authorities and there is ample experience and background data on this
species and strain.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Carboxy methyl cellulose 0.5% in aqueous solution

Details on exposure:

The test item was administered orally by gavage at a dose volume of 10mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered
to each animal on the basis of the most recently recorded body weight and the volume
administered was recorded for each animal.

Details on mating procedure:

Pairing was monogamous (one male to one female). A vaginal smear was taken from the
day after the start of pairing until positive identification of copulation (spermidentification,
vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.

Details on analytical verification of doses or concentrations:

Analysis was performed to confirmt hat the proposed formulation procedure was acceptable
and that the stability of the formulation was satisfactory (RTC Study No. A2386). In the
present study, samples of the formulations prepared on Weeks 1 and 7 of study were also
analysed to check the homogeneity and concentration. Results of analysis were within the
acceptability limits for suspension (with the exception of low dose level in the last week).

Since results of mid- and high dose levels were within the acceptability limits, the negative result pertaining the low dose level did
not affected the integrity of the study. Chemical analysis was carried out by the Analytical
Chemistry Department at RTC according to a validated method (RTC Study No. A2386). The
software used for this activity was the Empower® 2 Build No. 2154.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior
to pairing and thereafter through the day before necropsy, for a minimum of 28 days. Dose
volumes were adjusted once per week for each animal according to the last recorded body
weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior
to pairing and thereafter during pairing, post coitum and post partum periods until Day 13
post partum. Dose volumes were adjusted once per week for each animal according to the
last recorded body weight. During the gestation and post partum periods, dose volumes were
calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on
Days 1, 4, 7 and 13 post partum.

Frequency of treatment:

once a day, 7 days a week

Details on study schedule:

The purpose of this study was to provide information on toxicity as well as any possible
effects of Iodobis(triphenylphosphino)copper on male and female reproductive performance
such as gonadal function, mating behaviour, conception, development of conceptus and
parturition.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels of 100, 300 and 1000 mg/kg/day were selected in consultation with the
Sponsor based on information from a preliminary non-GLP compliant study (RTC Study no.
E0107). In this study, the test item was administered for 2 weeks at dose levels of 100, 300
and 1000 mg/kg bw/day in male and female rats. No clinical signs, no effects on body weight
and food consumption, no changes in organ weight and no treatment-related macroscopic
observation were noted in the treated animals, when compared to controls.

Positive control:

No

Parental animals: Observations and examinations:

Mortality
Throughout the study, all animals were checked early in each working day and again in
the afternoon. At weekends and Public Holidays a similar procedure was followed except
that the final check was carried out at approximately mid-day. This allowed post mortem
examinations to be carried out during the working period of that day.

Clinical signs
Once before commencement of treatment and at least once daily during the study, each
animal was observed and any clinical signs recorded. Observations were performed at the
same time interval each day, the interval was selected taking into consideration the presence
of post-dose reactions. All observations were recorded for individual animals.

Body weight
Males were weighed weekly from allocation to termination. Females were weighed weekly
from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20.
Dams were also weighed on Days 1, 4, 7 and 13 post partum and on the day of necropsy

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during
the pre-mating period starting from allocation. Individual food consumption for the females
was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Days 4,
7 and 13 post partum starting from Day 1 post partum.

Oestrous cyclicity (parental animals):

Vaginal smears and oestrous cycle
Females were evaluated for oestrous cyclicity at least 7 days before allocation to groups
and animals that exhibited irregular cycle were not included in the study. Vaginal smears
were also taken daily in the morning starting from allocation up to pairing until a positive
identification of copulation was made (data pertaining before allocation and before dosing
periods are not presented and archived with study raw data).
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken on the day of necropsy.

Litter observations:

Pups identification, weight and observation
As soon as possible after parturition was considered complete (Day 0 post partum), all pups
(live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum.
Pups humanely killed (2 male and 2 female pups from dam X0580007) or dying (one female
pup from dam X0580007 and one male pup from dam X0580013 in control group, two male
pups from dam X0580029 in low dose group and one male pup from dam X0580057 in
mid-dose group) during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters.

Adjustment of litter size (culling) and pups selection for blood collection at necropsy
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled
to 8 (4 males and 4 females, where possible) by a random selection. Partial adjustment was
acceptable. Two litters were culled to less than 8 pups: 2 males and 2 females from dam
X0580043, 3 males and 2 females from dam X0580047, in order to have at least 2 pups for
thyroid hormone evaluation.

Pup blood collection – Day 4 post partum

Litters with more than 8 pups
On Day 4 post partum, two pups (males or females, selected for culling) were sacrificed for
blood collection and the serum samples stored at -80°C. From dam X0580051 only one female
pup was culled and bled for thyroid hormone evaluation.

Litters with less than 8 pups
On Day 4 post partum for litters with less than 8 pups, two of the pups (preference was given
to females in order to retain more male pups for possible nipple retention on Day 14 post
partum) from the litter were sacrificed for blood collection and serum samples stored at -80°C.

Nipple count at Day 13 post partum
On Day 13 post partum, pups were observed to verify the presence of nipple areolas and the
results of observation were recorded.

Anogenital distance (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to a
measure of pup size, the cube root of body weight collected on Day 1 post partum.

Postmortem examinations (parental animals):

Parental males
The males were killed after the mating of all females, after at least 28 days of treatment period.

Parental females
The females with live pups were killed on Day 14 post partum.
In addition, female no X0580007 with total litter loss on Day 7, was killed on Day 14.

Necropsy and blood collection
The clinical history of adult animals was studied and a detailed post mortem examination
was conducted (including examination of the external surface and orifices). Changes were
noted, the requisite organs weighed and the required tissue samples preserved in fixative
and processed for histopathological examination.

All females were examined also for the following:
– number of visible implantation sites (pregnant animals)
– number of corpora lutea (pregnant animals)
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium
sulphide to reveal evidence of implantation.

Organ weights
Parental animals
From all animals completing the scheduled test period, the organs indicated in section 4.5.6
were dissected free of fat and weighed. The ratios of organ weight to body weight were
calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in section 4.5.6 were fixed and preserved in 10% neutral
buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides
which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed in section 4.5.6. After dehydration
and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer
thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides
were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological
evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was
performed initially in all animals in the control and high dose males killed at the end of the
study.
In the first instance, the examination was restricted as detailed below:
i Tissues specified in section 4.5.6 in all males and females from the control and high
dose groups killed at term.
ii All abnormalities in all groups.
The examination was extended to include the liver in the high dose and control females, and
thyroid in the high dose and control male pups sacrificed on Day 14 post partum, based on
the results of liver and thyroid weights in agreement with the Sponsor.

Postmortem examinations (offspring):

Pups
Pups killed for humane reasons or those that had completed the scheduled test period (Day 4
post partum or Day 14 post partum) were euthanised by intraperitoneal injection of Sodium
Thiopenthal.

All pups found dead in the cage or sacrificed for humane reasons were examined for external
and internal abnormalities.
All culled pups sacrificed at Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed at Day 14 post partum were examined for external abnormalities and
sex confirmation by gonads inspection.

Nipple retention at Day 14 post partum
No nipples/areolae were seen in male pups and no tissue was retained.

Blood collection
Blood samples were taken as detailed below:
On Day 14 post partum, approximately 0.5 mL of blood samples were taken from at least two
pups (1 sample/sex) per litter.

Organ weights
Thyroid was weighed from one male and one female pup selected for blood collection of hormones
determination and preserved in 10% neutral buffered formalin for histopathological
examination. The thyroid weight was determined after fixation.

Tissues fixed and preserved
Samples of all the tissues listed were fixed and preserved in 10% neutral
buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides
which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed. After dehydration
and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer
thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides
were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological
evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was
performed initially in all animals in the control and high dose males killed at the end of the
study.
In the first instance, the examination was restricted as detailed below:
i Tissues specified in all males and females from the control and high
dose groups killed at term.
ii All abnormalities in all groups.
The examination was extended to include the liver in the high dose and control females, and
thyroid in the high dose and control male pups sacrificed on Day 14 post partum, based on
the results of liver and thyroid weights in agreement with the Sponsor.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance
of the differences amongst group means were assessed by Dunnett’s test or a modified t
test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric
Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric
version of theWilliams test. The criterion for statistical significance was p<0.05.

Reproductive indices:

For males and females each:
Copulation Index
Fertility Index

For males and females
Copulatory interval = Mean number of days between pairing and mating

For females
Pre-implantation loss
Pre-birth loss on Day 0 post partum
Post-natal loss on Day 4 post partum
Post-natal loss on Day 13 post partum

Sex ratios were calculated at birth and on Day 4 and on Day 14 post partum and were
presented as the percentage of males per litter.

Offspring viability indices:

Pre-implantation loss
Pre-birth loss on Day 0 post partum
Post-natal loss on Day 4 post partum
Post-natal loss on Day 13 post partum

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hard abdomen was seen in treated animals at all dose levels, in a dose-related manner and
with a slightly higher incidence in females than in males. Swollen abdomen and salivation
were seen in one female receiving 300 mg/kg/day on occasion. The other clinical signs
(scab/hairloss/damaged tail) were considered of no toxicological significance, since they
were seen occasionally in single animals of intermediate groups.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect on body weight of males before pairing, during the mating phase up to
the final sacrifice. The body weight gain was significantly reduced in males receiving 300 and
1000 mg/kg/day on Day 8 of the premating period. However, this difference was likely due to
an excessive increase of body weight gain in the control group. No changes in body weight
or body weight gain were seen in females before pairing, during mating, gestation and post
partum periods.

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No significant changes were seen in parental males regarding serum thyroid hormones (T3
and T4) and TSH levels.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were noted. In addition, histomorphology of the liver was
comparable to control and high dose treated females. Seminiferous tubules were evaluated
with respect to their stage in the spermatogenic cycle and to the integrity of the various cell
types within the different stages; regular layering in the germinal epithelium was noted. The
sporadic lesions reported in control and treated animals were considered to be an expression
of spontaneous and/or incidental pathology, commonly seen in this species and age.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Measurement of oestrous cycle did not show relevant differences between groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The outcome of mating performance (the mean time between pairing and mating, copulatory
and fertility indices) did not show differences related to treatment.

No intergroup differences were seen in number of corpora lutea, implantation, litter size
at birth and gestation length which was of 22 days (mean value). Slight increase in preimplantation
and pre-birth losses were seen in high dose group (1000 mg/kg/day), compared
to controls.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

reproductive function (oestrous cycle)

reproductive performance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Small, cold to touch, apparently no food intake and pale appearance were seen in treated
and control pups, with higher incidence in controls.
Pups found dead, between Day 1 and 7 post partum, were seen in control, in the low and
mid- dose groups (2 pups in the control and low dose groups and 1 pup in the mid-dose
group); pups missing, between Day 1 and 8 post partum, were recorded in all groups (13 in
the control, 1 each in the low and mid-dose and 9 in the high dose groups).
In addition, small, pale and cold to touch were the signs recorded in pups of one control dam,
humanely killed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pups found dead, between Day 1 and 7 post partum, were seen in control, in the low and
mid- dose groups (2 pups in the control and low dose groups and 1 pup in the mid-dose
group); pups missing, between Day 1 and 8 post partum, were recorded in all groups (13 in
the control, 1 each in the low and mid-dose and 9 in the high dose groups).

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease of circulating thyroxine (T4) (-18%) was instead observed
in the high dose male pups (100 mg/kg/day) sacrificed at Day 14 of age, when compared to
controls. The TSH levels appeared higher in this group and likely reflect a feedback response
to the lower circulating thyroid hormone. However, no consistency on hormone variations
due to the high intersubject variability of T3 and TSH, and/or dose relation was seen in the
other groups. In addition, no changes were recorded in thyroid histopathology of the high
dose male pups.
Therefore, due to the low severity and the absence of other related changes, this finding was
not considered toxicologically relevant.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Slight but statistically increases in ano-genital distance were seen in pups of both sexes, on
Day 1 of age at all dose levels, statistically significant in the mid- (5% in males and 11% in
females) and high dose (4% in males and 5% in females) groups.
These differences were of minimal entity, not clearly dose-related and within the range of our
historical control data, therefore considered incidental findings.

No nipples were observed in male pups at all dose levels.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were detected in decedent pups during the lactation period (Day 1-7 post
partum):
– two male and two female pups from dam X0580007, control group, were humanely
killed due to poor health status (small, pale and cold to touch);
– one female pup from dam X0580007 and one male pup from dam X0580013 in control
group, two male pups from dam X0580029 in low dose group and one male pup from
dam X0580057 in mid-dose group were found dead.
No remarkable differences were noted at the macroscopic observations of pups sacrificed at
culling and at term, between groups.

Histopathological findings:

no effects observed

Description (incidence and severity):

No changes were noted in the thyroid gland of high dose and control male pups sacrificed on
Day 14 post partum.

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks on result:

not determinable because of methodological limitations

Remarks:

OECD 421 is not designed to determine NOAEL for F1 generation

Reproductive effects observed:

no

Conclusions:

No effects were observed in in vivo parameters of parental animals: no effects on body weight,
body weight gain and food consumption were noted at any dose level investigated. Hard
abdomen was seen in all groups and both sexes for a short period and then recovered.

Thyroid hormones (T3, T4 and TSH) evaluated in parental males did not show any changes
of toxicological relevance.

No remarkable differences were noted at post mortem examination including organ weights
and no treatment-related microscopic changes were observed in treated animals, when
compared to controls. The statistically significant increase of the liver weight (up tp 18%)
observed in the high dose females, was not considered of toxicological significance based
on the comparable histomorphology of the liver between control and high dose females.

Futhermore, no effects on the spermatogenic cycle were described.

No effects on sexual function and fertility or in developmental parameters and lactation
were observed at any of the dose levels investigated: no intergroup differences were seen
in oestrous cycle, pre-coital intervals, copulatory and fertility indices; no significant differences
were observed in the number of corpora lutea, implantations, total litter size, preimplantation
loss and pre-birth loss and in gestation length between control and treated
females; litter data at birth and on Days 1, 4 and 13 post partum and sex ratios were also
comparable between treated and control females.

There were neither treatment-related signs at clinical observation of pups, nor at necropsy of
deceased pups or those sacrificed after culling or at term.

Increases in the ano-genital distance were seen in male and female pups on Day 1 of age in
all groups, significant at the statistical analysis in the mid- (up to 11%) and high dose (up to
5%) groups. While this effect can be considered not adverse in males it can be considered
adverse in females (masculinisation). However, these changes were of minimal entity, not
clearly dose-related and within the range of our historical control data, therefore considered
incidental findings.

No nipples were observed, on Day 13 of age, in male pups at all dose levels.

Some changes inclusing decreases in thyroid weight and circulating T4 levels were found
in high dose pups sacrificed at Day 14 of age. However, not clear consistency of hormones
variations, dose and/or sex relation was noted to finally address an adverse effect of the test
compound on thyroid function. Moreover, the histomorphology of thyroid gland did not
show any changes in high dose male pups compared to controls.

Executive summary:

Based on the results obtained in this study the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be 1000 mg/kg/day for males and females.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

satisfying

Justification for classification or non-classification

On the basis of the available data, no classification into the hazard class "reproductive toxicity" according to (EU) No. 1272/2008 is required.