Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2018 to 22 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: Pale yellow crystalline powder
Purity/Composition: 99.32%
Test item storage : At room temperature. From 16 Jul 2018: protected from light.

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
Receipt:
On 09 May 2018, 40 male and 40 female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 6-7 week-old and weighed between 134 and 173 g and females were 6-7 week-old and weighed between 105 and 151 g.
A health inspection was performed before the initiation of dosing.

Animal Identification:
Prior to start of the treatment period, each allocated animal was identified using earmark and tattoo.
Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Selection, Assignment, Replacement, and Disposition of Animals:
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Housing:
On arrival and following the pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room(s) in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

Environmental Conditions:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 21 to 22°C with an actual daily mean relative humidity of 41 to 67%. A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Environmental Acclimation:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to commencement of dosing.

Food:
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment:
For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
1% Aqueous carboxymethyl cellulose
Details on exposure:
Dose Formulation and Analysis:
Preparation of Test Item:

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 hours after completion of the preparation of the formulation.
Details of the preparation and dispensing of the test item have been retained in the Study Records.
Test item dosing formulations were kept at room temperature until dosing. From 16 Jul 2018 onwards, dosing formulations were protected from light (see deviation in Appendix 7). If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Any residual volumes were discarded.

Vehicle:
Identification: 1% Aqueous carboxymethyl cellulose

Vehicle Components:
Identification: Water (Elix)
Supplier: Millipore, Molsheim, France

Identification: Carboxymethyl cellulose
Supplier: Fagron, Capelle aan de IJssel, The Netherlands



Details on mating procedure:
On arrival and following the pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).

During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.

During locomotor activity monitoring, F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room(s) in which the animals were kept was documented in the study records.

Animals were separated during designated procedures/activities.

Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method:
Analyses were performed using a validated analytical procedure (Ardena Bioanalytical Laboratory AWI 4059, entitled: Irgacure TPO in formulations using LC-DAD, validated in Ardena Bioanalytical Laboratory validation study no. 15222).

Concentration Analysis:
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

Homogeneity Analysis:
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability Analysis:
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 510162, Ardena Bioanalytical Laboratory No. 15223) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions (room temperature) at the concentration range 10-100 mg/mL. Stability data have been retained in the study records of No. 510162.
As in the current study, the highest concentration of 120 mg/mL (Group 4) was outside the concentration range of 10-100 mg/mL tested before, stability of Group 4 formulation (120 mg/mL) was determined at 5 hours at room temperature under both normal laboratory light conditions and protected from light (to cover the first part of the treatment period during which formulations were not protected from light).
Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males per dose, 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Administration of Test Materials:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 12 weeks. Males were treated for 85-92 days, up to and including the day before scheduled necropsy. This included a minimum of 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating period. Females that delivered were treated for 113-127 days, i.e. 10 weeks prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 20 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 99-117 days.

The first day of dosing was designated as Day 1.

Female nos. 50 (Group 1), 51, 58 (Group 2), 60, 61, 62, 63, 64, 66 (Group 3) were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.

The dosing formulations were stirred continuously during dose administration.

A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.


Justification of Route and Dose Levels:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

The dose levels were selected based on the results of a 90-day repeated dose toxicity study with oral administration of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide in Wistar rats (ECHA dossier CAS number 75980-60-8) (data on file at Sponsor site), and in an attempt to produce graded responses to the test item.

In this 90-day study, a dose level of 1000 mg/kg resulted in clear toxic effects. Two out of ten females died at 1000 mg/kg and a reduced general health state of females was observed at this high-dose level. Body weight was also reduced in males at 300 mg/kg (10%) and in both males and females at 1000 mg/kg (23 and 8%, respectively). Signs of liver dysfunction together with an increase in liver and kidney weights were also observed in females and males at 1000 mg/kg. A reduction of testes size (on average by 50%) together with a marked diffuse atrophy of the testicular parenchyma and a slight to moderate interstitial edema were detected in males at 300 and 1000 mg/kg.

Based on these results and with the attempt to cause clear reproductive toxicity effects on males, a maximum level of 600 mg/kg was selected for the current study.

An elongated pre-mating period of 10 weeks (covering at least one spermatogenic cycle) was included in this study as adverse effects on the testes are expected.





Parental animals: Observations and examinations:
Mortality/Moribundity Checks – F0-Generation:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F0-Generation:
Clinical observations were performed at least once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
During the dosing period, these observations were performed at least after dosing.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body Weights – F0-Generation:
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 13 and 21 (see deviation in Appendix 7).
A terminal weight was recorded on the day of scheduled necropsy.

Food Consumption – F0-Generation:
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 13 and 21.

Water Consumption – F0-Generation:
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

Functional Tests – F0-Generation:
Functional tests were performed on the selected 5 males and females during the last week of the premating period (i.e. week 10 of treatment) . These tests were performed after completion of clinical observations.
The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

Cohabitation/Mating Procedure – F0-Generation:
After 10 weeks of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males. Since less than 9 females in Group 4 showed evidence of mating, each non-mated female was re-mated once with a male of proven fertility of the same group for a maximum of 7 days.

General Reproduction Data – F0-Generation:
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 was considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
Oestrous cyclicity (parental animals):
Estrous cycle determination – F0-Generation:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to the mating period, and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)
Clinical signs of preterm decedents are summarized in Mortality section 9.2.1.

Treatment-related clinical signs were noted during daily detailed clinical observations.

At 600 mg/kg, transient signs of abnormal behaviour and/or posture were noted during the premating period in all males and one female. Towards the end of Week 8 of treatment (premating period), all males at 600 mg/kg were noted less reactive (slightly calm/lethargic). This finding lasted for 1 week and was observed when opening the cage (before dosing), and persisted for a few minutes5. One female at 600 mg/kg (no. 79) was observed on three different occasions during Week 10-11 of treatment (end of the premating period-beginning of the mating period) with transient muscle twitching in combination with hunched posture and/or piloerection on 1 or 2 occasions (directly after dosing, after handling for performing the vaginal smear or in the cage just before handling). These clinical signs were short in duration (lasting for only a few minutes), and afterwards the female completely recovered(6). Piloerection was also noted in this female no. 79 and in two other females treated at 600 mg/kg (nos. 73 and 74) for 2 to 3 consecutive days at the end of Week 13 or 16 of treatment, respectively.

After the second week of the mating period, female no. 79 at 600 mg/kg was additionally noted with a transient reddish liquid secretion from and around the vagina(b). At the incidence observed, this clinical sign was considered a chance finding.

Salivation was observed in one control female on a single day, in several animals at 200 mg/kg and among all animals at 600 mg/kg for multiple days during the premating period (with a higher incidence and/or persistence in females) and mating period (only noted incidentally in a few males). This finding was considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Several animals among the groups were noted with alopecia, scabs and/or wounds and other incidental findings noted included rales and reddening of the ear. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered signs of no toxicological relevance.

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post-coitum and lactation phase.

(a) Time of occurrence and duration of these clinical signs were recorded in the Animal Daybook and are not included in the Clinical Signs Data Tables.
(b) This observation of animal no. 79 was recorded in the Animal Daybook and is not included in the Clinical Signs Data Tables.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
(Appendix 1 and Appendix 2)

There were 2 preterm decedents over the study period: one control female and one female at 600 mg/kg (both during the premating period).

One female of the 600 mg/kg group (no. 75) was sacrificed in extremis for animal welfare reasons, on Day 61 (Week 9 of the premating period). This female was noted with moderate lethargy, flat/hunched posture, muscle twitching, piloerection, slight chromodacryorrhoea, slight ptosis, and red snout. She presented with normal body weight gain from start of treatment onwards, with a slight body weight loss (2%) over Weeks 7-8 of the premating period, followed by recovery in Week 9. During the macroscopic examination at necropsy, accentuated lobular pattern of liver and reduced spleen were noted. Although no definite cause of moribundity could be established from the microscopic examination of the selected tissues, a relationship to treatment could not be excluded as comparable clinical signs were noted for a surviving high dose female as well (no. 79).

The other decedent was regarded to be unrelated to treatment with the test item: one control female (no. 45) was found dead before dosing on Day 43 (beginning Week 7 of the premating period). No relevant clinical signs were noted for this female from the start of the study period. During the macroscopic examination at necropsy, watery-clear content was observed in the thoracic cavity. Although no definite cause of death could be established during microscopic examination of the selected tissues, the macroscopic observation at necropsy of watery-clear content in the thoracic cavity could be indicative for a technical error during the oral gavage procedure.

One female of the 200 mg/kg group (no. 67) was euthanized on lactation Day 4, as she had a total litter loss.

Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)
A dose-dependent test item-related effect on body weight and body weight gain was observed in males at 200 and 600 mg/kg during both the premating and mating period.

Males treated at 600 mg/kg had a slightly reduced mean body weight gain from start of treatment onwards (reaching statistical significance on Day 8 and from Day 57 onwards), resulting in a 13% lower mean body weight when compared with control values at the end of treatment.

A slight reduction (non-significant) in mean body weight and body weight gain was also observed in males at 200 mg/kg from Day 29 (Week 5 of the premating period) onwards, that was mainly attributed to 2 animals in this group (nos. 22 and 29) that presented with lower body weight gain. At necropsy, a statistically significant decrease in mean body weight (9%) was observed at 200 mg/kg vs mean control.

No toxicologically relevant changes in body weight or body weight gain were observed in males treated at 60 mg/kg and females treated up to 600 mg/kg over the entire treatment period.

The lower body weight gain noted in females at 600 mg/kg during the gestation period was considered to be related to the non-pregnancy status of all the females at this high dose level, and as such not to reflect a systemic toxic effect of the test item (see Individual Data Tables).

The slightly reduced mean body weight and body weight gain observed at 200 mg/kg during the last phase of the gestation period was mainly attributed to one animal in this group (no. 68) that presented with only resorptions.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)
No toxicologically relevant changes in food consumption (before or after correction for body weight) were recorded over the treatment period in all treated animals of both sexes.
The lower mean value for absolute food consumption recorded in females at 600 mg/kg from Day 14 post-coitum onwards, was considered to be related to their non-pregnancy status. After allowance for body weight, food consumption remained within normal range (see Individual Data Tables).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2)

Thyroid hormone analyses:
Serum levels of T4 in F0-males were considered not to be affected by treatment up to 600 mg/kg.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)

Functional tests were performed at the end of the 10-week premating period for all groups (5 animals/sex/group) to further investigate the abnormal behaviour and muscle twitching observed during clinical observations in Group 4 animals towards the end of the premating period.

Hearing ability, pupillary reflex, static righting reflex, and grip strengths of fore and hind limb were considered to be unaffected by treatment up to 600 mg/kg.

For treated males, a decrease in fore and hind limb grip strength was observed, reaching statistical significance for fore limb grip strength in all treated groups. However, as all values remained well within the available historical control range for male rats of this strain and age(a), and no dose-related trend could be established, this finding was considered unrelated to treatment. It should be noted that mean control value for fore limb grip strength was at the upper limit of the historical control data. This could explain the statistically significant changes seen in treated males. Also in females, fore and hind limb grip strength was considered to be unaffected by treatment as all mean values remained within the normal range for female rats of this strain and age(a).

A dose-dependent decrease of total movements and ambulations was noted in females, but changes did not reach statistical significance and all mean values remained within the available range of historical control range(b). Therefore, no toxicological relevance was attached to this finding. Also in males, a slight decrease in motor activity (non-significant) was observed at 600 mg/kg but mean values remained within the normal range(b). Although, this decrease was considered not toxicologically relevant, a relationship with treatment could not be discarded based on the clinical signs observed at this dose level.

(a) Grip Strength Historical Control Data in Wistar Han rats (2013-2018):
Fore leg (male): mean = 1073, P5-P95 = 623-1676 (N = 199).
Hind leg (male): mean = 690, P5-P95 = 368-999 (N = 199).
Fore leg (female): mean = 893, P5-P95 = 525-1324 (N = 198).
Hind leg (female): mean = 529, P5-P95 = 310-758 (N = 208).

(b)Motor Activity Historical Control Data in Wistar Han rats (2012-2018):
Total movements (male): mean = 3648, P5-P95 = 2123-5617 (N = 369).
Ambulations (male): mean = 733, P5-P95 = 305-1198 (N = 369).
Total movements (female): mean = 4858, P5-P95 = 2561-8187 (N = 379).
Ambulations (female): mean = 1328, P5-P95 = 629-2281 (N = 369).

Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)

Test item-related lower testes and epididymides weights (absolute and relative to body weights) were noted in the 600 mg/kg treated males as shown in text table 10.

Text Table 10
Mean Percent Organ Weight Differences from Control Groups

Males
Dose level (mg/kg/day): 60 200 600

TESTES
Absolute 0 4 -52**
Relative to body weight 4 15* -45**

EPIDIDYMIDES
Absolute -2 -11* -43**
Relative to body weight 1 -1 -34**
*: P<0.05, **: P<0.01

There was a decrease in mean epididymis weight (absolute) at 200 mg/kg which was normalized after correction for terminal body weight.

Mean organ:body weight ratios of thyroid and seminal vesicles weights were statistically significantly higher in males at 600 mg/kg. These organ weight differences were statistically significant when compared with the control group but were considered to be the result of a test item-related effect on final body weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)
Macroscopic observations at necropsy revealed test item-related alterations in the reproductive organs of males at 600 mg/kg: macroscopic findings were present in the testes as flaccid (8/10 animals) and reduced in size (10/10 animals) and in the epididymides as reduced in size (9/10 animals).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one female in each group including controls, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(Appendix 5)
Test item-related microscopic findings after treatment with Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide were noted in the thyroid gland, testes and epididymides and are summarized in text tables 11 and 12.

Text Table 11
Summary Test Item-Related Microscopic Findings – Males and Females
Males Females
Dose level (mg/kg/day): 0 60 200 600 0 60 200 600

THYROID GLANDSa 10 10 10 10 9 10 10 10
Hypertrophy follicular cell
Minimal 3 5 2 5 5 3 3 4
Slight - - 4 3 - - - 2
Colloid alteration
Minimal - - 3 2 - - - 2
Slight - - - 4 - - - 1
a = Number of tissues examined from each group.

Thyroid gland:
Hypertrophy follicular cell was present at increased incidence and severity in males treated at 200 and 600 mg/kg and in females treated at 600 mg/kg up to slight degree.
Colloid alteration was present in males treated at 200 and 600 mg/kg and in females treated at 600 mg/kg up to slight degree.


Text Table 12
Summary Test Item-Related Microscopic Findings – Males
Males
Dose level (mg/kg/day): 0 60 200 600

TESTES a 10 10 10 10
Atrophy tubular
Massive - - - 10
Atypical residual bodies
Slight - - 9 -
Moderate - - 1 -
Multinucleated giant cells
Moderate - - 1 1
Degeneration germ cells
Moderate - - 1 -
Depletion germ cells
Moderate - - 1 -

EPIDIDYMIDESa 10 10 10 10
Cell debris
Minimal - - - 2
Slight - - - 5
Moderate - - 1 1
Reduced sperm
Slight - - 1 -
Massive - - - 10
a = Number of tissues examined from each group.

Testes:
Tubular atrophy was present in all males treated at 600 mg/kg at massive degree. This correlated with the decreased testes weight and with the macroscopic finding flaccid and/or reduced in size.
Atypical residual bodies were present in all males treated at 200 mg/kg at slight to moderate degree.
Multinucleated giant cells were present in a single male treated at 200 mg/kg and in a single male treated at 600 mg/kg at moderate degree.
Degeneration and depletion germ cell were present in a single male treated at 200 mg/kg at moderate degree.

Epididymides:
Cell debris was present in a single male treated at 200 mg/kg at moderate degree and in most males treated at 600 mg/kg up to moderate degree.
Reduced sperm was present in a single male treated at 200 mg/kg at slight degree and in all males treated at 600 mg/kg at massive degree. This correlated with the decreased epididymides weight and with the macroscopic finding of reduced in size.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Reproductive performance

Text Table 13
Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups
Group Dose level
mg/kg bw/day Female/Male nos. In-Life Reason Histopathology
1 0 - - -
2 60 56/16 Not pregnant -
3 200 67/27 Total litter loss -
68/28 Implantation sites only Testes: moderate degeneration and depletion germ cells
4 600 72/32 and 36 No evidence of mating Testes: massive tubular atrophy Epididymides: massive reduced sperm
74/34 and 38 No evidence of mating
79/39 and 40 No evidence of mating
71/31 Not pregnant
73/33 Not pregnant
76/36 Not pregnant
77/37 Not pregnant
78/38 Not pregnant
80/40 Not pregnant

There were 9/9 couples treated at 600 mg/kg, compared to 2/10 at 200 mg/kg and 1/10 at 60 mg/kg, that failed to deliver healthy pups. The males treated at 600 mg/kg all showed massive tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. The lack of offspring for one couple treated at 200 mg/kg could be explained by the moderate depletion and degeneration of sperm cells with multinucleated giant cells in the testes and moderate cell debris and slight reduced sperm in the epididymides. The rest of the males treated at 200 mg/kg all showed atypical residual bodies, which apparently did not affect their fertility.

There were no morphological findings in the reproductive organs of the females which could be attributed to the test item and stage aware evaluation of the testes of males treated at 60 mg/kg did not show any indication for abnormal spermatogenesis.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
(Appendix 2)
Length and regularity of the estrous cycle were not considered to be affected by treatment up to 200 mg/kg.

All females had regular cycles of 4 days. Extended di-estrus during the mating period occurred in one female at 60 mg/kg (no. 53) and 3 females at 600 mg/kg (nos. 72, 74 and 79). An extended di-estrus occasionally occurs at low incidence in untreated controls. Therefore, and due to the absence of a dose-related incidence at 200 mg/kg and based on the fact that this low dose female had a normal litter, the single occurrence at 60 mg/kg was considered a chance finding. However, for the three high dose females with irregular cycle during the mating period a relation to treatment with the test item could not be excluded. For all three females mating could not be confirmed, even though they had been cohoused for another 7 days, after 14 days of unsuccessful mating, with a male of the same group for which mating was already confirmed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating Index:
(Appendix 1 and Appendix 2)
Mating index was considered to be affected by treatment at 600 mg/kg: 3 out of 9 high dose females (nos. 72, 74 and 79) showed no evidence of mating after a prolonged cohabitation period of a total of 21 days with two different males.
For all females at 60 and 200 mg/kg mating could be confirmed.
The mating indices were 67% at 600 mg/kg and 100% for all the other groups. Mating index at 600 mg/kg was below the 5th percentile of the historical control range (mean=99%, P5-P95=90-100%, N=98).

Precoital Time:
(Appendix 1 and Appendix 2)
Precoital time was considered not to be affected by treatment up to 600 mg/kg for all mated females. The majority of these females showed evidence of mating within 4 days.
One control female (no. 41) and one low dose female (no. 60) had a precoital time of 5-6 days. Another low dose female (no. 53, presenting with extended di-estrus) had a precoital time of 14 days. In the absence of a dose-related response, this increase in the precoital time was considered unrelated to treatment.

Number of Implantation Sites:
(Appendix 1 and Appendix 2)
At 600 mg/kg, all 6 mated females presented with 0 implantation sites (and 0 corpora lutea).
At 60 and 200 mg/kg, mean number of implantation sites remained in the same range of controls. All mean values were within the historical control range(10). However, at the individual level, there was one female at 200 mg/kg (no. 68) with only 5 implantation sites. Such low numbers are occasionally seen in females of this strain and age, but as the lack of offspring for this animal could be explained by the effects observed on the male sperm (moderate depletion and degeneration of sperm cells with multinucleated giant cells in the testes and moderate cell debris and slight reduced sperm in the epididymides), it was considered to be related to treatment.

Fertility Index:
(Appendix 1 and Appendix 2)
Fertility index was considered to be affected by treatment at 600 mg/kg: all 6 mated females in the high dose group were not pregnant (0 implantation sites).
All mated females at 60 and 200 mg/kg were pregnant, except for one low dose female (no. 56). As this was an isolated finding in the low dose group only, it was considered to be unrelated to treatment.
The fertility indices were 100%, 90%, 100% and 0% for the control, 60, 200 and 600 mg/kg groups, respectively.

(10) Historical Control Data in Wistar Han rats (2015-2018):
Number of implantation sites: mean = 12.3, P5-P95 = 6-16 (N = 920).

Gestation Index and Duration:
(Appendix 1 and Appendix 2)
Gestation index and duration of gestation were considered not to be affected by treatment up to 200 mg/kg.
Except for one female at 200 mg/kg (no. 68) with implantation sites only, all pregnant females had live offspring. The gestation indices were 100%, 100% and 90% for the control, 60 and 200 mg/kg groups, respectively.

Parturition/Maternal Care:
No signs of difficult or prolonged parturition were noted among the pregnant females by treatment up to 200 mg/kg.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Litter Size:
(Appendix 1 and Appendix 2)
Litter size was considered not to be affected by treatment up to 200 mg/kg.
Live litter sizes were 10.6, 10.8 and 10.4 living fetuses/litter for the control, 60 and 200 mg/kg groups, respectively. One female (no. 67) at 200 mg/kg had only 1 living pup.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 2)
No clinical signs occurred among pups that were considered to be related to treatment.
For the 2 pups of control litter no. 42 which were found dead at first litter check on PND 1, and for the pup of litter no. 53 (at 60 mg/kg) which was found dead on PND 2, absence of milk in the stomach was noted at first litter check.
One pup from litter no. 62 (at 200 mg/kg) was noted with a (little) swollen circle at the tail apex over lactation Days 13-22. The nature and incidence of this and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Live Birth Index:
(Appendix 1 and Appendix 2)
The number of live offspring on Day 1 after littering compared with the total number of offspring born was considered not to be affected by treatment up to 200 mg/kg.
Live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 97% for the control and 99% for the 60 and 200 mg/kg groups.
Two pups of the control group (no. 42) were found dead at first litter check. As the control group was treated with the vehicle alone, a relation to treatment with the test item could be excluded.

Viability Index:
(Appendix 1 and Appendix 2)
The number of live offspring on Day 4 before culling compared with the number of offspring on Day 1 was considered unaffected by treatment up to 200 mg/kg.
Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) were 99% for all the groups.
One control pup (litter no. 43) and one pup at 60 mg/kg (litter no. 53) were missing or found dead at PND 2. In addition, one female (no. 67) at 200 mg/kg had lost her single pup on
PND 4. The pups missing were most likely cannibalised. Lobuloalveolar development was observed in the mammary gland of animal no. 67 which could not explain the total litter loss in this female. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2)
Body weights of pups were considered not to be affected by treatment up to 200 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)
The number of live offspring on Day 20 after littering compared to the number of live offspring on Day 4 (after culling) was considered not affected by treatment. No pups were found dead/missing between lactation Days 5 and 20, resulting in a lactation index of 100% for all groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2)
Serum T4 levels in male and female PND 21-23 pups were considered not to be affected by treatment up to 200 mg/kg.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
No milk in the stomach was noted for the 2 pups of control litter no. 42 which were found dead at first litter check on PND 1. As the control group was treated with the vehicle alone, a relation to treatment with the test item could be excluded.
Description (incidence and severity):
Note: As female no. 56 at 60 mg/kg was not pregnant, developmental data are available of only 9 females in the low dose group. As all females at 600 mg/kg were not mated or non-pregnant, no developmental data were available for evaluation in the high dose group.

Post-Implantation Survival Index:
(Appendix 1 and Appendix 2)
The total number of offspring born compared to the total number of uterine implantations was considered unaffected by treatment up to 200 mg/kg.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94%, 90% and 85% for the control, 60 and 200 mg/kg groups, respectively. The slightly lower post-implantation survival index observed at 200 mg/kg was mainly attributed to a single female in this group (no. 67) that had only 1 living pup out of 6 implantation sites. At the isolated incidence, and as all mean values were within the historical control range (mean=92%, P5-P95=83-99%, N=98), no toxicological relevance was attached to this finding.
For control female no. 44, the number of pups (11) was slightly higher than the number of implantations (10). This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.

Sex Ratio:
(Appendix 1 and Appendix 2)
At 60 and 200 mg/kg, sex ratio (% of males/females) was reduced (43/57 and 41/59, respectively) when compared with concurrent controls (57/43), reaching statistical significance at 200 mg/kg. This decrease in the % of living males was mainly attributed to single females at 60 mg/kg (no. 52) and 200 mg/kg (no. 67) that presented with a low % of males/females. Therefore, this finding was considered unrelated to treatment.

Anogenital Distance:
(Appendix 1 and Appendix 2)
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered to be unaffected by treatment up to 200 mg/kg.

Areola/Nipple Retention:
(Appendix 1 and Appendix 2)
Treatment up to 200 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No developmental toxicity was observed by treatment up to 200 mg/kg. At 600 mg/kg, no litters were available for evaluation due to the non-pregnancy status of females.
Critical effects observed:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Dose Formulation Analyses

(Appendix 4)

Accuracy

The concentrations analyzed in the formulations of Groups 2, 3 and 4 (samples prepared for use on 04 June 2018; Week 3) and Group 4 (samples prepared for use on 07 September 2018;  Week 17) were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

The analytical run performed by Ardena Bioanalytical Laboratory on formulation samples prepared in Week 1 (16 May 2018) was not accepted since procedural recovery samples were outside the acceptance criteria.  Therefore, a second occasion for formulation sample collection and analysis of all groups was included in Week 3 (04 June 2018).

No test item was detected in the Group 1 formulation.

Homogeneity

The formulations of Group 2 and Group 4 (samples prepared for use on 04 June 2018; Week 3) were homogeneous (i.e. coefficient of variation ≤10%).

Stability

Formulations of Group 4  (samples prepared for use on 07 September 2018; Week 17) were stable when stored either at room temperature under normal laboratory light conditions or protected from light for at least 5 hours (i.e. relative difference ≤10%).

Conclusions:
In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide were established:

Parental NOAEL: 200 mg/kg (Based on clinical signs and reduced body weight gain (males).)

Reproduction NOAEL: 60 mg/kg (Based on adverse microscopic findings in the male reproductive organs at 200 and 600 mg/kg.)
(Based on the low mating and fertility index at 600 mg/kg.)

Developmental NOAEL: 200 mg/kg (no data available at 600 mg/kg).

Executive summary:

The objectives of this study were to determine the potential toxic effects of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide when given orally by gavage for a minimum of 12 weeks to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.  An elongated pre-mating period of 10 weeks was included, to cover at least one complete spermatogenic cycle.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 60, 200, and 600 mg/kg/day, based on the results of a 90-day repeated dose toxicity study with oral administration of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide in Wistar rats (ECHA dossier CAS number 75980-60-8) (data on file at Sponsor site).

The study design was as follows:

Text Table 1

Experimental Design

Group No. Test Item Identification Dose Level (mg/kg/day) Dose Volume (mL/kg) Dose Concentration (mg/mL) Number of Animals
Males Females
1 - 0 (Vehicle) 5 0 10 10
2 Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide 60 5 12 10 10
3 200 5 40 10 10
4 600 5 120 10 10

Chemical analyses of formulations were conducted during the study to assess accuracy, homogeneity, and stability (for Group 4) over 5 hours.

The following parameters and end points were evaluated in this study:  mortality/ moribundity, clinical signs, functional observations1 (for 5 selected animals/sex/group), body weight and food consumption, estrous cycle determination, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined:  mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 21-23 pups)).

Formulation analyses confirmed that formulations of test item in 1% Aqueous carboxymethyl cellulose were prepared accurately and homogenously.  The stability analysis performed showed that the high concentration formulation was prepared accurately and was stable for 5 hours at room temperature, both protected from light and under normal laboratory conditions.

Parental results

There were 2 preterm decedents over the study period.

At 600 mg/kg, one female was sacrificed in extremis for animal welfare reasons, on Day 61 (Week 9 of the premating period) as she presented with moderate lethargy, flat/hunched posture, muscle twitching, piloerection, slight chromodacryorrhoea, slight ptosis, and red snout.  Slight (2%) body weight loss was noted for this female over Weeks 7-8 of the premating period, followed by recovery in Week 9.  During the macroscopic examination at necropsy, accentuated lobular pattern of liver and reduced size of the spleen were noted.  Although no definite cause of moribundity could be established from the microscopic examination of the selected tissues, a relationship to treatment could not be excluded as comparable clinical signs were noted for a surviving high dose female as well.

The other preterm decedent (one control female during the premating period) was regarded to be unrelated to treatment with the test item.  In addition, one female at 200 mg/kg was euthanized on PND4 as she had a total litter loss during the lactation period.

Treatment-related clinical signs were noted in males and females at 600 mg/kg at the end of the premating period.

At 600 mg/kg, transient signs of abnormal behaviour and/or posture were noted towards the end of the premating period in all males and two females (including the preterm sacrificed female).  During 7 days at the end of Week 8 of treatment, all males at 600 mg/kg were noted less reactive (slightly calm/lethargic).  At the incidence observed, this clinical sign was considered to be related to treatment.  In addition, one female at 600 mg/kg was observed on three different occasions during Week 10-11 of treatment with transient muscle twitching in combination with hunched posture and/or piloerection on 1 or 2 occasions.  These clinical signs were short in duration (lasting for only a few minutes), followed by complete recovery.  As similar clinical signs were also noted in the high dose female sacrificed in extremis, these observations were regarded as related to treatment.  In addition, piloerection was also noted in this female and two other females treated at 600 mg/kg for 2 to 3 consecutive days at the end of Week 13 or 16 of treatment.

Test item-related effects on body weight and body weight gain were observed in males at 200 and 600 mg/kg during both the premating and mating period.  High dose males presented with a slightly reduced mean body weight gain from start of treatment onwards (reaching statistical significance on Day 8 and from Day 57 onwards), resulting in a 13% lower mean body weight at the end of treatment when compared with control values.

The reduced mean body weight observed in males at 200 mg/kg at the end of the treatment period was considered non-adverse, based on the slight magnitude of the change (less than 10%) and as no other relevant clinical signs were noted at this dose level.

The lower body weight gain and absolute food consumption noted in females at 600 mg/kg during the gestation period was considered to be related to the non-pregnancy status of all the females at this high dose level, and as such not to reflect a systemic toxic effect of the test item.

A dose-dependent decrease of total movements and ambulations was noted in females, but changes did not reach statistical significance and all mean values remained within the available range of historical control range.  Therefore, no toxicological relevance was attached to this finding.  Also in males, a slight decrease in motor activity (non-significant) was observed at 600 mg/kg but mean values remained within the normal range.  Although, this decrease was considered not toxicologically relevant, a relationship with treatment could not be discarded based on the clinical signs observed at this dose level.

Macroscopic observations at necropsy revealed test item-related alterations in the reproductive organs of males at 600 mg/kg:  macroscopic findings were present in the testes as flaccid (8/10 animals) and reduced in size (10/10 animals) and in the epididymides as reduced in size (9/10 animals).

A test item-related decrease in organ weights of testes and epididymides (absolute and relative to body weights) were noted in males at 600 mg/kg.  Differences vs control in organ:body weight ratio were 45% and 34% for testes and epididymis, respectively.

Adverse test item-related microscopic findings were noted in the testes and epididymides in males starting at 200 mg/kg.  Relationships were observed between gross necropsy, organ weight, and histopathology observations.

The massive tubular atrophy observed in testes at 600 mg/kg was considered adverse.  In addition, the atypical residual bodies in males at 200 mg/kg and the single male with degeneration/depletion of germ cells at 200 mg/kg were also considered adverse.  In the epididymides; the massive reduced sperm at 600 mg/kg and the slight reduced sperm at 200 mg/kg with minimal to moderate cell debris were considered adverse.

Non-adverse test item-related microscopic findings were noted in the thyroid gland in males at 200 and 600 mg/kg and in females at 600 mg/kg.  The minimal increase in hypertrophy of the follicular epithelium of the thyroid glands and the colloid alteration seen in males starting at 200 mg/kg and in females at 600 mg/kg was considered non-adverse at current severities and in absence of any other adverse pathologic findings.

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. food consumption and male T4 thyroid hormone).

Reproductive results

Reproduction toxicity was observed at 200 and 600 mg/kg.

At 600 mg/kg, mating index (67%) was lower when compared with concurrent control (100%) and mean historical control value (99%).  During the mating period, extended di-estrus was observed in the 3 high dose females that were not mated.  Although an extended di-estrus occasionally occurs at low incidence in untreated controls, a relation to treatment with the test item could not be excluded.

At 600 mg/kg, the fertility index was 0%.  There were 9/9 couples treated at 600 mg/kg, compared to 2/10 at 200 mg/kg and 1/10 at 60 mg/kg, that failed to deliver healthy pups.  All the males treated at 600 mg/kg showed massive tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring.  The lack of offspring for one couple treated at 200 mg/kg (female with only implantations) could be explained by the moderate depletion and degeneration of sperm cells with multinucleated giant cells in the testes and moderate cell debris and slight reduced sperm in the epididymides.  The rest of the males treated at 200 mg/kg all showed atypical residual bodies, which apparently did not affect their fertility.

No treatment-related changes were noted in the precoital time.

Developmental results

No developmental toxicity was observed by treatment up to 200 mg/kg.  At 600 mg/kg, no litters were available for evaluation due to the non-pregnancy status of females.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, maternal care and postnatal pup development consisting of mortality, clinical signs, body weight, sex ratio, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide were established:

Parental NOAEL:              200 mg/kg (Based on clinical signs and reduced body weight gain (males).)

Reproduction NOAEL:       60 mg/kg (Based on adverse microscopic findings in the male reproductive organs at 200 and 600 mg/kg.)

(Based on the low mating and fertility index at 600 mg/kg.)

Developmental NOAEL:       200 mg/kg (no data available at 600 mg/kg).

1Functional tests were performed at the end of the 10-week premating period for all groups (5 animals/sex/group) to further investigate the abnormal behaviour and muscle twitching observed during clinical observations in Group 4 animals towards the end of the premating period.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
(Jan. 2001)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
(May 2008)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
(August 1998)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 to 14 weeks
- Weight at study initiation:
- Housing: individually in Macrolon plastic cages (MIII type)
- Diet (e.g. ad libitum): ad lib. (SM R/M-Z from SSNIFF)
- Water (e.g. ad libitum): ad lib. (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10 aph
- Photoperiod (hrs dark / hrs light): 12h / 12h
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 10, 30, 100 mg/ml
- Amount of vehicle (if gavage): 5ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 12 October 2015). The samples were dispatched on dry ice to ABL where they were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 5 hours at room temperature (lowest and highest concentration).

- No test item was detected in the Group 1 formulations.
- The concentrations analyzed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).
- The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
- Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).


Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor and based on data from a maternal toxicity study, (BASF Project 10R0490/03X053, WIL Project 510610). In this study, the pregnant female rats treated at 500 mg/kg bw/day showed reduced body weight gain arising from reduced food consumption. 5/6 animals showed hunched posture, piloerection and/or salivation and increased liver weights were apparent at termination. No clear signs of toxicity were observed in pregnant female rats treated at 150 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative assessment was introduced.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 post-coitum (or within 24h of abortion or early delivery)
- Organs examined: All macroscopic abnormalities (external, thoracic, abdominal) were recorded, collected and fixed in 10% buffered formalin
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 and distribution of implantations: Yes (In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites )
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: sex and weight of each fetus, weight of each placenta (of live fetuses only)
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter, i.e., the fetuses also used for visceral examination]
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 6) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 7(many-to-one rank test)) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 8) was applied to frequency data.
- The Mann Whitney test (Ref. 9) was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 10) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Ref. 11) was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss = (number of corpora lutea - number of implantation sites) / number of corpora lutea
Post-implantation loss = (number of implantation sites - number of live fetuses) / number of implantation sites
Viable fetuses affected/litter = number of viable fetuses affected/litter / number of viable fetuses/litter
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Five females delivered early on Day 21 post-coitum (one vehicle control, one 50 mg/kg bw/day treated female, two 150 mg/kg bw/day treated females and one 500 mg/kg bw/day treated female). Additionally, one female in the 500 mg/kg bw/day group had an early delivery on Day 20 post-coitum.

The incidence and persistence of salivation showed a dose related increase. In 14/22 females treated with 150 mg/kg bw/day salivation was observed for one to a few days at the end of treatment. For one to several days, salivation was noted in 19/22 females in the highest dose group (500 mg/kg bw/day). Since no correlated findings were noted, this was attributed to the taste of the test item and of no toxicological relevance.

Furthermore, piloerection in 7/22 females and hunched posture in 4/22 females was observed for one to several days in females treated at 500 mg/kg bw.day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain was significantly reduced in the highest dose group compared to vehicle controls from Day 9 post-coitum onwards. Consequently, on Day 21 post-coitum mean body weights and weight gain corrected for uterus weight were significantly lower in 500 mg/kg bw/day treated females compared to the controls (mean body weight on day 21 post coitum was 285 g compared to 305 g in controls). Body weights and body weight gain were unaffected at 50 and 150 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the beginning of treatment (Day 6 to Day 12 post-coitum), food consumption was significantly reduced in Group 4 compared the control Group (maximum approximately 20%). This fully recovered to similar levels as controls from Day 12 post-coitum onwards. Food consumption was unaffected at 50 and 150 mg/kg bw/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No macroscopic findings were noted that were considered to be treatment related.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no effects on the number of pregnant females, corpora lutea, implantation site and pre- or post-implantation loss noted with treatment up to 500 mg/kg bw/ day.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects on the number of pregnant females, corpora lutea, implantation site and pre- or post-implantation loss noted with treatment up to 500 mg/kg bw/ day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no effects on the number of pregnant females, corpora lutea, implantation site and pre- or post-implantation loss noted with treatment up to 500 mg/kg bw/ day.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Four animals were found not pregnant, (two 50 mg/kg bw/day treated females, one 150 mg/kg bw/day treated female and one 500 mg/kg bw/day treated female). All other females were pregnant and had litters with viable fetuses.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day, female fetal weights were slightly, but significantly lower compared to the control group (4.8 gram versus 5.1 gram in controls). A similar, but not significant, reduction was observed in male fetal weights (5.0 gram versus 5.3 gram in controls) and consequently combined fetal weights.
The placenta weights were unaffected by treatment up to 500 mg/kg bw/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 500 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was unaffected by treatment up to 500 mg/kg bw/day. The numbers of fetuses (litters) available for morphological examination on Day 21 post-coitum were 233 (22), 200 (20), 202 (21) and 202 (20) in Groups 1, 2, 3, and 4, respectively.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 500 mg/kg bw/day. The only external malformations observed, were noted in two fetuses from a litter at 500 mg/kg bw/day. Fetus A086-05 had no tail and fetus A086-06 had a tail that was filamentous, which was confirmed at skeletal examination. Both these tail malformations were not seen previously among historical controls, however, because they pertain to the same group of tail abnormalities and occurred in one litter, they can be considered genetic in origin and are not related to treatment.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was an increase in the number of fetuses with bent limb bones in the highest dose group. Ten fetuses from 5 litters were affected with this skeletal malformation, compared to one control fetus (bent limb bones were not observed at 50 and 150 mg/kg bw/day). In all cases one or both scapulae were bent and in three fetuses also humeri were involved. The incidence of bent limb bones at 500 mg/kg bw/day was far above the upper limit in historical control fetuses (10.6% versus 0.7% per litter, respectively) and therefore the bent limb bones were considered to be treatment related. The higher incidence of bent limb bones at 500 mg/kg bw/day coincided with an increased litter incidence for bent ribs in this group. Mean litter proportions for this skeletal variation were 13.5%, 23.5%, 22.1% and 69.9% per litter in Group 1, 2, 3 and 4, respectively. The incidence in Group 4 was statistically significantly increased and all fetuses with bent limb bones also had bent ribs.

At 500 mg/kg bw/day, the incidence of the skeletal variations reduced ossification of the skull and unossified metatarsals and metacarpals were significantly increased compared to the control group. Mean litter incidences for reduced ossification of skull bones were 12.4%, 12.5%, 21.1%, 45.9% and for unossified metatarsals and metacarpals 5.4%, 6.6%, 3.2% and 21.0% per litter in the control, 50, 150 and 500 mg/kg bw/day group, respectively. Both these parameters are indicative of retarded skeletal ossification which is in line with the lower fetal body weights in this dose group. Both findings are considered to be related to the decreased maternal body weights (-7%).
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 500 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: forelimb
skeletal: hindlimb
Description (incidence and severity):
See above
Developmental effects observed:
not specified

In the abscence of gross limb malformations, and in the presence of retardation as a consequence of maternal toxicity, bent limb bones could be considered temporary variations rather than malformations. It is hypothesized, that during development the increase in muscle mass puts stress on the bones. If ossification is delayed, the bones might not be able to counteract this pressure and appear bowed until ossification is finalized. Bone development in rats continues long after birth, extending into young adulthood. In a few studies, pups were followed sequentially after birth, and bent long bones and scapulae were transient in nature and appeared normal by the time of weaning.

References:

- De Schaepdrijver L., Delille P., Geys H., Boehringer-Shahidi C., Vanhove C. In vivo longitudinal micro-CT study of bent long limb bones in rat offspring. Reprod Toxicol 46, 91–97 (2014).

- Mitchard T.L., French J. Apparent postnatal recovery of chondrodystrophy in the Harlan Han Wistar rat. Reprod Toxicol 32, 169–70 (2011).

- Kimmel C.A., Garry M.R., DeSesso J.M. Review Articel: Relationship Between Bent Long Bones, Bent Scapulae, and Wavy Ribs: Malformations or Variations? Birth Defects Research (Part B) 101:379–392 (2014)

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Dec 2017 to 25 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Appearance: Pale yellow crystalline powder
Purity/Composition: 99.32%
Test item storage: At room temperature

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Justification for Test System and Number of Animals:
The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch (DEC 14-49 incl. amendment(s)) as required by the Dutch Act on Animal Experimentation (February 1997).

Animal Identification:
At study assignment, each animal was identified by tattoo in the ear.

Environmental Acclimation:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 2 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals:
On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 25% of the mean per subgroup. Females which were mated on the same day were classified in the same subgroup.

Husbandry:
Housing:
On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles. The rooms in which the animals were kept was documented in the study records.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number.

Environmental Conditions:
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 19 to 20°C with an actual daily mean relative humidity of 68 to 73%. (see deviation in Appendix 9). A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food:
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap water was freely available to each animal via water bottles/containers.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment:
For psychological/environmental enrichment, animals were provided with shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands).

Veterinary Care:
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
1% Aqueous carboxymethyl cellulose
Details on exposure:
Preparation of Test Item:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 hours after adding the vehicle to the test item.
Details of the preparation and dispensing of the test item have been retained in the Study Records.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Any residual volumes were discarded.

Food:
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water:
Municipal tap water was freely available to each animal via water bottles/containers.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Vehicle(s):
Identification: 1% Aqueous carboxymethyl cellulose

Vehicle Components:
Identification: Water, Supplier: Elix, Millipore S.A.S., Molsheim, France
Identification: Carboxymethyl cellulose, Supplier: Fagron, Capelle aan de IJssel, The Netherlands
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample Collection and Analysis:
Dose formulation samples were collected for analysis as indicated:


Dose Formulation Sample Collection Schedule:

Occasion: Week 1 of treatment: 18 Dec 2017
Concentration: All groups
Homogeneity: Groups 2 and 4(a)
Stability: Groups 2 and 4

Occasion: Week 4 of treatment: 10 Jan 2018
Concentration: Group 2
Homogeneity: -
Stability: -

(a)The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.

Stability samples were kept at room temperature under normal laboratory light conditions for 5 hours, and then placed on dry ice. All other samples were stored on dry ice immediately after sampling. All samples to be analyzed were shipped on dry ice to ABL BV on the following dates: 19 Dec 2017 and 10 Jan 2018. The analytical laboratory was notified before shipment of the samples. Upon receipt at the analytical laboratory, the samples were stored in the freezer ≤ -70 °C until analysis.
Based on the results of the Week 1, additional Group 2 samples collected in Week 4 of treatment. However, in consultation with the Sponsor and after re-evaluation of the results samples were not analyzed. Samples were stored at -70°C for a maximum of 37 days (available long term stability information) and will be discarded after the expiry date.
Residual samples were discarded after completion of the sample analysis.

Analytical Method:
Analyses were performed by using a validated analytical procedure (ABL No. 15222; Test Facility Study No. 510160).

Concentration Analysis:
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.

Homogeneity Analysis:
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.

Stability Analysis:
During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined for 5 hours at room temperature.
Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Duration of treatment / exposure:
Administration of Test Materials:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe.

The dosing formulations were stirred continuously during dose administration.

A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Frequency of treatment:
7 days a week from Day 6 to Day 28 post-coitum, inclusive.
Duration of test:
29 days
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females per dose level.
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of Route and Dose Levels:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of the dose range finder (Test Facility Study No. 20134097, see Appendix 7), and in an attempt to produce graded responses to the test item.

Selection, Assignment, Replacement, and Disposition of Animals:
On the day of receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 25% of the mean per subgroup. Females which were mated on the same day were classified in the same subgroup.
Maternal examinations:
In-life Procedures, Observations, and Measurements – F0-Generation:
The in-life procedures, observations, and measurements listed below were performed for parental animals.

Mortality/Moribundity Checks – F0-Generation:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail.

Clinical Observations – F0-Generation:
Clinical observations were performed at least once daily, beginning on Day 6 post-coitum and lasting up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Cage debris was examined to detect abortion or premature birth.

Body Weights – F0-Generation:
Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

Food Consumption – F0-Generation:
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

Water Consumption – F0-Generation:
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Terminal Procedures – F0-Generation:
Terminal procedures are summarized in the following table.

Text Table 4
Terminal Procedures

Group No. No. of Females Scheduled Euthanasia Day Necropsy Procedures Histology and Histopathology
Necropsy Collection of Gross Lesions Organ Weights
1 22 29 post-coitum X X X -
2 22 -
3 22 -
4 22 -
Unscheduled Deaths X X - -

X = Procedure to be conducted; - = Not applicable.

Unscheduled Deaths – F0-Generation:
If necessary for humane reasons, animals were euthanized as per Test Facility SOPs. These animals were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%), underwent necropsy, and specified tissues were retained.

Scheduled Euthanasia – F0-Generation:
Animals surviving until scheduled euthanasia were euthanized by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol® 20%). No body weight were recorded at necropsy.
Scheduled necropsy was conducted on the following days:

Females surviving to planned necropsy: Day 29 post-coitum.
Females with early delivery: (no. 66, 85, 87, 88): Within 24 hours of early delivery.


Ovaries and uterine content:
Necropsy – F0-Generation:
All animals (including animals sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the uterus) were weighed.
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy, except for female no. 49).
• The number and distribution of live and dead fetuses.
• The number and distribution of embryo-fetal deaths.
For animals sacrificed before planned necropsy, these findings were reported in the individual data tables only.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
Fetal examinations:
Terminal Procedures – F1-Generation:
Method of Euthanasia – F1-Generation:
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube.
Fetuses of animals sacrificed before planned necropsy were externally examined in detail and euthanized by sodium pentobarbital.

Fetal Examinations – F1-Generation:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External Examinations – F1-Generation:
Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. Weights were not determined for fetuses of animals sacrificed before planned necropsy or females with early delivery, except for fetuses of female no. 49 (sacrificed on Day 28 post-coitum).
For late resorptions and recognizable fetuses of females euthanized in extremis, a gross external examination was performed.

Visceral Examinations– F1-Generation:
All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar2.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique3. After examination, the tissues without variation or malformations were discarded. Selected tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

Skeletal Examinations– F1-Generation:
All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson4.
Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Historical control data:
Historical control data for New Zealand White (Crl:KBL (NZW)) rabbits (period 2012-2017; n=495):
- Corrected body weight gain (gram): Mean: -245; p5 - p95: -557.3 – 45.4
- Corrected body weight gain (%): Mean: -6; p5 - p95: -13.8 - 1.3
- Weight of the uterus (gram): Mean: 524; p5 - p95: 360.3 - 699.8
See also Appendix 3, attached below
Clinical signs:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2)
No treatment-related clinical signs were noted up to 100 mg/kg.
Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
(Appendix 1 and Appendix 2)
No mortality occurred during the study period that was considered to be related to treatment with the test item.
Two females from the mid dose group (30 mg/kg) were euthanized prematurely. Female no. 64 was euthanized on Day 26 post-coitum, as she was found lethargic with hunched posture, piloerection and ptosis. Additionally, reduced faeces production, faeces containing mucus and red fluid were observed on the manure tray. From post-coitum Day 21 onwards, this female had consumed no food and showed slight body weight loss. At necropsy, intussusception of the caecum with dark red discolouration was noted, which correlated with the poor clinical status of this female. Based on the single occurrence of this finding, it was considered a chance finding and unrelated to treatment.
Female no. 49 was euthanized on Day 28 post-coitum, as she experienced labored respiration on two subsequent days and gasping on one day. At necropsy, the left caudal lobe of the lungs was found perforated. Additionally, a reddish contents of the trachea, many dark red foci on the lungs and a reddish, watery-clear fluid in the thoracic cavity were noted. Taken together, these findings were indicative of a dosing related incident and the preterm sacrifice of this female was considered unrelated to the test item.
Four females (one at 30 mg/kg and three at 100 mg/kg) were sacrificed prematurely after an early delivery, for details see maternal pregnancy data)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2)
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
Body weight gain corrected for weight of the gravid uterus was slightly lower in 100 mg/kg treated females compared to the concurrent controls. However, as the effect was minimal, not statistically significant, and remained within the historical control range it was considered unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2)
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 100 mg/kg.
Any statistically significant changes in food consumption were considered to be unrelated to treatment since no trend was apparent regarding dose.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings among control and treated animals included emaciated appearance, cysts of the ovaries or oviducts, alopecia, scabs or scars. These findings are occasionally seen among rabbits used in these types of study and in the absence of a dose relationship were considered unrelated to treatment.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
Note: In order to enter female numbers into WTDMSTM an adjustment in the numbering was made, for example: female 1 was reassigned as female A001, female 2 as A002 etc. Also numbering of fetuses was changed; fetus 1 of female 1 was reassigned as A001-01 etc.
Overall, the number of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and treatment groups were similar and in the range of normal biological variation.
One female (no. 66) treated at 30 mg/kg and three females (nos. 85, 87 and 88) treated at 100 mg/kg delivered early on Day 27 or 28 post-coitum. These females had relatively low to no food consumption in the six days prior to the early delivery. Except for cannibalism of two fetuses (one at 30 mg/kg and one at 100 mg/kg), no external abnormalities were observed for the premature litters.
Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
necropsy findings
Abnormalities:
effects observed, non-treatment-related
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 100 mg/kg.
The combined fetal weights in the 100 mg/kg dose group was slightly decreased compared to the concurrent controls. However, as the decrease was only slight (5% lower than concurrent controls), in the absence of corroborative findings of growth retardation at skeletal examination (see 9.4.3) and without statistical significance, this finding was considered not toxicologically relevant.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
The male:female ratio was unaffected by treatment up to 100 mg/kg.
Mean sex ratios (males:females) were 48:52, 48:52, 49:51 and 46:54 for the control, 10, 30 and 100 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 9.1, 9.1, 9.1 and 9.5 fetuses/litter for the control, 10, 30 and 100 mg/kg groups, respectively.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
There were no treatment related effects on external morphology following treatment up to 100 mg/kg.
External malformations were observed in two, three and one fetus(es) of respectively the control, 10 and 30 mg/kg groups, but not at 100 mg/kg. Carpal and/or tarsal flexures (all without apparent skeletal origin) occurred in the 30 mg/kg fetus (A048-09) and two control fetuses (A007-01 and A008-04) of which fetus A008-04 also had an omphalocele and multiple malformations viscerally. An omphalocele was also observed in 10 mg/kg fetus A038-11 and the other externally affected fetuses at 10 mg/kg either had cyclopia (fetus A029-03) or meningocele (fetus A030-02).
As the above malformations occurred singly in the 10 and 30 mg/kg groups, not at 100 mg/kg and/or were seen in concurrent and historical control fetuses (except for cyclopia), they were considered chance findings and were not related to treatment.
External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
A statistically significant increase in the incidence of malaligned sternebra(e) was observed at 100 mg/kg; 9.2% per litter compared to 3.8% for the concurrent controls. The value at 100 mg/kg remained within the maximum value of the available historical control data (10.2% per litter).
All remaining variations noted, were not considered treatment related as they occurred infrequently and/or at frequencies that were within the range of available historical control data.
Only one skeletal malformation was observed in this study, a vertebral anomaly with associated rib anomaly, and as it occurred in two control fetuses only (A004-10 and A013-04), it was as such considered to be spontaneous in origin.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
(Appendix 1 and Appendix 2 (Historical Control Data is reported in Appendix 3)).
There were no treatment related effects on visceral morphology following treatment up to 100 mg/kg.
Visceral malformations occurred in 2 (2), 3 (3), 2 (2) and 1 (1) fetuses (litters) in the control, 10, 30 and 100 mg/kg groups, respectively. At 100 mg/kg, fetus A078-07 had abnormal lobation of the liver, while at 30 mg/kg, fetus A058-11 had both kidneys and testes malpositioned. Malpositioning of kidneys and/or testes were also observed in 30 mg/kg fetus A057-04 (one kidney), 10 mg/kg fetus A036-10 (both kidneys and testes) and control fetus A008-04 (both testes). In fetus A036-10 this was found together with an intestine diverticulum and the affected control fetus had multiple other malformations, namely interrupted aortic arch, liver anomaly, anomalous content of kidneys and ureters and hemorrhagic eyes.
The two remaining viscerally malformed fetuses were 10 mg/kg fetus A029-03, which had transposition of the great vessels and control fetus A013-05 with a narrow aorta and ventricular septum defect.
The very low incidence and group distribution of these malformations does not indicate a treatment relationship. Moreover, all but two of the malformations observed in test item treated groups (transposition of the great vessels and intestine diverticulum) were noted previously in historical controls.
All the variations noted, were considered unrelated to treatment as they occurred infrequently, in the absence of a dose-related incidence trend and/or occurred at frequencies that were within the range of available historical control data.
Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
skeletal malformations
visceral malformations
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
no

Dose Formulation Analyses

(Appendix 5)

No test item was detected in the Group 1 formulation.

The concentrations analyzed in the formulations of Groups 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).

The concentration of Group 2 had a mean accuracy of 83.4% and was therefore not in agreement with the target concentration.  

The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).

As the mean accuracy of the Group 2 was only minimally below specifications and the homogeneity for the formulations was acceptable, the accuracy of preparation of the Group 2 formulations was considered acceptable.

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide was established as being at least 100 mg/kg.

Higher doses were not tolerated as 3/6 females treated at 200 mg/kg in the Dose Range Finder were sacrificed in extremis (see Appendix 7).
Executive summary:

The objectives of this study were to determine the potential of Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive.  In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg, based on the results of the dose range finder (Test Facility Study No. 20134097, see Appendix 7).

The study design was as follows:

Text Table 1

Experimental Design

Group No. Test Item Identification Dose Level (mg/kg/day)a Dose Volume (mL/kg) Dose Concentration (mg/mL) Number of Females
1 - 0 (Control) 5 0 22
2 Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide 10 5 2 22
3 Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide 30 5 6 22
4 Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide 100 5 20 22

aThe test item has a purity of 99.32%, dose calculations were not corrected for purity.

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.

The following parameters and end points were evaluated in this study for the F0-generation:  mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.  

In addition, the following parameters were determined for the F1-generation:  the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.  Test formulations prepared were considered stable, for at least 5 hours at room temperature.

At 30 and 100 mg/kg, respectively one and three females delivered their litters prematurely on Day 27 or 28 post-coitum.  Except for cannibalism of two fetuses (one at 30 mg/kg and one at 100 mg/kg), no external abnormalities were observed for the premature litters.  These females had relatively low to no food consumption in the six days prior to the early delivery which probably caused these early deliveries.  In absence of any toxicity up to 100 mg/kg, these early deliveries were regarded as not toxicologically significant.  

No treatment-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. mortality, clinical appearance, body weight, food consumption and macroscopic examination).

No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. fetal body weights, external, visceral (including sex) and skeletal malformations and developmental variations.

A treatment related increase in the incidence of malaligned sternabra(e) was observed at 100 mg/kg; 9.3% per litter compared to 3.8% in the concurrent controls.  However as malaligned sternebra(e) are defined as skeletal variation and the observed incidence remained within the historical control maximum value (10.2% per litter), this finding was considered non-adverse.

In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide of  at least 100 mg/kg was established.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

The previous lead registrant submitted an OECD 414 conducted in the rat. This found developmental effects (skeletal) on pups. The 28 day and 90 day repeat dose toxicity studies also noted a reduction in the weight of male reproductive organs (testes), but did not record observations on sperm or fertility. based upon this limited information the current harmonised classification was set at Repr. Toxicity 2 (H360).

The results of this second species (rabbits) study did not corroborate the adverse prenatal developmental toxicity data seen in the first species (rats).  Therefore, no changes to the prenatal developmental toxicity classification were proposed.  The highest test dose (100 mg/kg bw/day) in the rabbit was lower than that used in the rat OECD 414 (500 mg/kg bw/day) and rat OECD 421 (600 mg/kg bw/day) studies due to the tolerability (toxicity) of the substance in rabbits being lower than in rats.This difference in maternal/parental toxicity may be the cause of the absence of developmental effects and should be taken into consideration when assessing whether or not a mechanistic mode of action applies between the species.

The effects seen on the testes and epididymides in rats clearly result in lower levels of fertility, which fulfil the criteria for Repro. Cat. 1B., and there is no evidence that raises doubt on the relevance of this effect for humans.