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REACH

2-amino-2-methylpropanol

EC number: 204-709-8 | CAS number: 124-68-5

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

A) ORAL ROUTE

I. Selection of key animal oral studies for long-term systemic DNEL:

15 animal studies are available on the repeated dose oral toxicity of AMP (including via reproduction and developmental toxicity studies) so the AMP toxicological database is of unprecedented quality and allows a highly robust assessment. Studies are outlined in Table 1 below. AMP was tested under multiple forms with variable pH (as highly alkaline AMP, pH-adjusted AMP, or AMP HCl where pH is also artificially reduced), variable purity (industrial or high-purity grade) and dosing regimen (in diet or by gavage), in multiple species (rat, mouse, rabbit, dog, monkey) and over multiple dosing periods (5 days to 1 year). NB: Clinical data are also available and summarized in next sections.

The liver was AMP's target organ in all animal studies which included suitable investigations. Generally, this was evidenced by increased liver enzymes and liver weight and hepatocyte vacuolation. Only in two dog studies were irreversible liver effects noted (hepatocyte necrosis and fibrosis). The only other toxic effect was local toxicity caused by the alkalinity of the test material, in studies with no or partial pH adjustment. There was no clear trend concerning compared sensitivity between males and females.

Table 1: Overview of animal repeat-dose toxicity studies

Study N°

Reference

Species

Duration (weeks)

AMP grade tested

AMP form tested

Dosage form pH at LOAEL, if AMP hadn't been neutralized

Gives robust information about liver toxicity ?

Irreversible liver toxicity observed ?

NOAEL males

NOAEL females

LOAEL males

LOAEL females

MTD males

MTD females

Pittz 1977

Rat

0.7 (5d)

Industrial

As is

>11 at all doses

No (liver histopathology after 10-day recovery)

1000 to <2500

500 to <1000

Carney 2005

Rat

High-purity

Neutralized

No LOAEL

No (no liver histopathology)

not reached

Carney 2005

Rat

5 to 8

High-purity

Neutralized

10.6-10.7

Yes

213

<71

710

not reached

Goldenthal 1976

Rat

Industrial

Neutralized ?

11.2-11.7

Yes

162

345

314

717

not reached

717 to <1355

Lankas 1981

Rat

Industrial

Neutralized

10.8-11.1

Partial (liver histopathology at top-dose only)

187

233

not reached

Pittz 1977/1979

Rat

Industrial

As is

>11 at all doses

No (no liver histopathology)

< 500

500 to <750

Pittz 1977/1979

Rat

Industrial

Neutralized

12.3

Partial (liver histopathology at top-dose only)

<=750

<=1100

not reached

Millard 2021

Rat

High-purity

Neutralized

10.9-11.1

Yes

>=142

142

not reached

Goldenthal 1976

Dog

Industrial

Neutralized ?

11.0

Yes

Scattered hepatocyte necrosis, slight fibrosis

54 to <62

70 to <108

Lankas 1981

Dog

Industrial

Neutralized

11.0-11.2

Yes

Very slight scattered hepatocyte necrosis, moderate fibrosis

not reached

Griffin 1990/1993

Dog

Industrial

Neutralized

No LOAEL

No (too low doses tested)

>=2.8

>=2.7

not reached

Griffin 1990/1993

Dog

Industrial

Neutralized

No LOAEL

No (too low doses tested)

>=2.7

>=2.4

not reached

Pittz 1977

Monkey

0.7 (5d)

Industrial

As is

>11 at all doses

No (liver histopathology after 10-day recovery)

< 500

Murphy 2020

Rabbit

Industrial

Neutralized

11.7 (at lethal dose)

No (no liver histopathology)

23.7 to <71.0

Goldenthal 1976

Mouse

Industrial

Neutralized ?

No LOAEL

No (too low doses tested)

>=501

>=685

not reached

Red: exclusion criteria for use to derive a long-term DNEL. Bold: key studies. ND: not determined due to missing investigations. All NOAEL/LOAEL/MTD values are in mg AMP/kg bw/day.

Following selection criteria were used to select the two key studies to derive a long-term DNEL:

studies shorter than 4 weeks are excluded;

studies which failed to identify a LOAEL (too low dose-levels) are excluded;

studies which did not investigate liver histopathology at all dose-levels are excluded, as liver is the target organ and histopathology the best way to determine adversity of effects;

as above exclusion criteria pre-select 5 robust studies in two species (rats: 5-8, 8 and 18 weeks; dogs: 4 and 14 weeks), the study of longest duration in rat and dog is selected.

As a consequence:

for rats, the key study is Millard's 2021 18-week study (OECD 443 added with liver histopathology).

for dogs, the key study is Lankas' 1981 14-week study (similar to OECD 409).

both studies involved neutralization of AMP's pH and are thus largely over-conservative. This is further discussed below.

II. Species sensitivity ranking by oral route:

Studies are available in mouse, rat, rabbit, dog, monkey and human. We can attempt to rank their relative sensitivity for systemic toxicity (survival and liver histopathology) based on inter-species comparison of NOAEL and LOAEL values for similar exposure durations:

human cannot be ranked: low drug doses so no mortality and no liver toxicity observed (see details in next sections).

concerning general toxicity based on comparison of MTD values:
- rabbits are more sensitive than monkeys over 5 days to 3 weeks: lethality was reached at 1000 mg AMP/kg bw/day in monkeys although pH was >11 (study N° 13), and as low as 71.0 mg AMP/kg bw/day in rabbits although pH was neutralized (study N° 14);
- rabbits are more sensitive than dogs over 3-4 weeks (studies N° 9 and 14);
- monkeys are more sensitive than rats over 5 days (studies N° 1 and 13);
- dogs are more sensitive than rats over 4-8 weeks (studies N° 3, 4 and 9);
- rats are more sensitive than mice over 8 weeks (studies N° 4 and 15).

concerning liver toxicity based on comparison of NOAEL-LOAEL ranges:

dogs are more sensitive than rats over 4-8 weeks (studies N° 3, 4 and 9) and 14-18 weeks (studies N° 8 and 10);

rats are more sensitive than mice over 5-8 weeks (studies N° 3, 4 and 15).

Therefore the sensitivity ranking is:

general toxicity: rabbit > monkey/dog > rat > mouse (insufficient data to compare dog/monkey and to rank humans)

liver toxicity: dog > rat > mouse (insufficient data to rank rabbit, monkey and humans)

III. Animal oral key studies on AMP-HCl:

A) Rat 18-week study (Millard, 2021):

In an OECD 443 study, groups of 19 to 30 CD rats/sex/dose were fed diets containing AMP-HCl over two generations. Toxicologically relevant effects were limited to liver toxicity in male rats of F0 and F1A generations (doses converted into AMP):

At 142 mg AMP/kg bw/day, liver weights were 12% increased in F0 only, and minimal to moderate (adverse) hepatocellular vacuolation was seen in 24/30 F0 rats and 8/20 F1A rats;

At 71 mg AMP/kg bw/day, non-adverse hepatocellular vacuolation was seen in 11/25 F0 rats (minimal to mild) and 7/22 F1A rats (minimal);

At 35 mg AMP/kg bw/day, non-adverse minimal hepatocellular vacuolation was seen in 4/25 F0 rats and 1/22 F1A rats.

The NOAEL for parental/general toxicity was 71 and 142 mg AMP/kg bw/day in males and females, resp. The absence of irreversible liver lesions means effects do not warrant STOT RE classification.

At the tested dietary concentrations, pH of non-neutralized AMP solutions would range 10.4-11.1. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is instead of its neutralized form AMP-HCl, not even the study's low-dose could have been tested due to dose-limiting, pH-mediated toxicity and no other toxic effects would have been noted. Since OECD guidelines do NOT require any neutralization or pH adjustment, this study represents artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity. This bias introduced by neutralisation is discussed in more details under below section "Additional information".

B) Dog 14-week study (Lankas, 1981):

In a 14-week repeated-dose study similar to OECD 409, groups of 4 beagle dogs/sex/dose were fed diets containing AMP-HCl. Following effects were noted (doses converted into AMP):

At 81 mg AMP/kg bw/day: Lower body weight gain and hemoglobin concentration. Multiple indications of liver toxicity: higher serum liver enzyme activities, lower cholesterol, increased liver weights, discoloration and mottling of liver, microscopic lesions including vacuolization, periportal cirrhosis (lipid deposition, hepatocellular necrosis and fibrosis) and bile duct hyperplasia. Also some local irritation: minimal to moderate chronic gastritis.

At 17 mg AMP/kg bw/day: Non-adverse liver effects (increased weight, minimal to moderate reversible lesions) and local irritation (gastric inflammation).

At 0.78 (males)/0.85 (females) mg AMP/kg bw/day: local irritation (gastric inflammation).

The systemic NOAEL was 17 mg AMP/kg bw/day in both sexes. Applicability of STOT RE classification is discussed under below section "Justification for classification or non-classification".

At the tested dietary concentratioins, pH of non-neutralized AMP solutions would range 10.1-11.1. Thus, as the OECD 443 study, this study represents artificial and non-guideline dose maximization by orders of magnitude, removing of AMP's critical toxic effect, which is pH-dependent non-specific toxicity. This bias introduced by neutralization is discussed in more details under below section "Additional information".

IV. Human oral key studies on pamabrom (see Exposure related observations in humans, IUCLID § 7.10.3):

Pamabrom is an Over The Counter (OTC) diuretic agent which is an AMP salt of 8-bromotheophylline, CAS No. 606-04-2.
It is an equimolar mixture of 74.4% w/w 8-Bromotheophylline (CAS No. 10381-75-6) and 25.6% w/w 2-amino-2-methylpropan-1-ol (AMP, CAS No. 124-68-5). Clinical data on this test material can be used for AMP safety assessment based on the AMP/Pamabrom bioequivalence study (see Basic Toxicokinetics /IUCLID §7.1.1) which indicated that Pamabrom's AMP, and AMP as is, were equivalent in terms of AUC0-t, AUC0-inf and AUC0-168h. Pamabrom doses can be converted into equivalent AMP doses based on the AMP content of 25.6% w/w in this drug and assuming a patient weight of 70 kg when not indicated.

A) Clinical trials:

Six clinical studies are available on pamabrom. NOAELs are expressed in AMP based on pamabrom posology and AMP content. In all cases the below-listed NOAELs were the maximum tested dose:

1) Trial in severe cardiac failure patients (Doherty et al, 1953): Only 2/18 patients reported adverse effects potentially related to pamabrom upon longer treatment: 1/18 case of maculopapular rash (4th week of treatment) and 1/18 case of diarrhea (day 120 of treatment). A third patient stopped treatment after 2 weeks due to therapeutic success. The NOAEL was therefore >= 2.2 mg AMP/kg bw/day x 2 to 17 weeks depending on patient.

2) Trial in women with premenstrual tension - Preliminary study (McGavack et al, 1956): No adverse effect in n=9 women. The NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks.

3) Trial in women with premenstrual tension - Main study (McGavack et al, 1956): No adverse effect in n=43 women. The NOAEL was >= 1.5 mg AMP/kg bw/day x 2 weeks (mean: variable
treatment regime).

4) Trial in women with primary dysmenorrhea (Ortiz et al, 2016): Minor effects in 3/189 patients (somnolence, dizziness and increased thirst, 1 case each). The NOAEL was >= 0.32 mg AMP/kg bw/day x 3 days.

5) Trial in pregnant women (Patterson, 1958): No adverse effects in n=38 pregnant women with edema classified as mild pre-eclampsia. The NOAEL was >= 5.9 mg AMP/kg bw/day x 1 week (if considering only the second treatment cycle), and >= 2.9 mg AMP/kg bw/day x 2 weeks (if considering both treatment cycles in patients treated twice).

6) Trial in pregnant women (James et al, 1957): No adverse effects in n=180 pregnant women with edema treated for unknown duration (Klimisch 4 study). The max. NOAEL was >= 2.2 mg AMP/kg bw/day, and in most patients it was >=1.6 mg AMP/kg bw/day.

Considering all clinical trials, the most robust human NOAEL for repeat-dose toxicity comes from study 2) with monitoring of hematology and blood biochemistry. The NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks.

B) 70 years of safe clinical use - Pharmacovigilance argument:

In the US, pamabrom is used since early 1950's so it has a track-record of >70 years of safe use in an ill population. Based on the National Library of Medicines database, at least 20 US OTC drugs currently contain pamabrom (full list provided in study summary). Their claims include back/leg/joint pain and edema (1 drug) and premenstrual and menstrual pain/discumfort/edema (19 drugs). Based on contents and posology for each drug, AMP has a human NOAEL of >=0.73 mg AMP/kg/day for up to 10-day treatment cycles, repeated over periods.

C) Overall human oral NOAEL for repeat-dose toxicity:

No serious adverse effect was ever reported during 70 years of clinical trials and medication with pamabrom based on 5 publications (6 trials) and posology of 20 OTC drugs. The most robust human NOAEL for repeat-dose toxicity comes from study 2) (preliminary study by McGavack et al, 1956) where the NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks. All other studies and OTC drug posology confirm AMP NOAEL values in the mg/kg bw/day range, always the highest tested dose whatever the treatment duration (3 days to 17 weeks + one trial where treatment duration was not indicated).

V. Selection of final study for human long-term oral DNEL calculation:

Key NOAEL values are available in rats, dogs and humans as summarized below and each may in principle be used to derive a long-term human DNEL. However, interim calculations (simplified calculations not down to the actual DNEL value) are sufficient to demonstrate that a single NOAEL value can be selected to derive the long-term human DNEL values:

Species	Study	Test item	Study duration (weeks)	Assessment factor for extrapolation to chronic exposure (1)	Systemic NOAEL in most sensitive sex (mg AMP/kg bw/day)	Actual body weight at NOAEL, week 1 to last study week, mean	Allometric scaling factor to a 70kg-human, based on mean actual body weight (ECHA Guidance R.8, 2008) (2)	Remaining interspecies variability	Intraspecies variability (for general population)	Corrected NOAEL (4) equivalent to a long-term systemic oral DNEL for general population
Rat	Millard, 2021	AMP-HCl	18	1.5 (1)	71 (males)	443g to 717g, mean: 580g (males)	3.31	2.5	10	= 71/1.5/3.31/2.5/10 = 0.57 mg AMP/kg bw/day
Dog	Lankas, 1981	AMP-HCl	14	2	17 (both sexes)	8.5kg to 10.6kg, mean 9.55kg (males) 8.4kg to 9.9kg, mean 9.15kg (females) overall mean: 9.35 kg	1.65	1.5 (3)	10	= 17/2/1.65/1.5/10 = 0.34 mg AMP/kg bw/day
Human	McGavack, 1956 (preliminary study)	pamabrom	4	6	>= 2.9 (women); (5)	Not indicated	1	1	3 (6)	>= 2.9/6/1/1/3 = 0.16 mg AMP/kg bw/day (5)

(1): lower assessment factor used for 18-week study as the default value of 2 applies to a 13-week study, and AMP has no bioaccumulation potential

(2): = (BWhuman/BWanimal)^0.25

(3): lower assessment factor used as the default value of 2.5 in when only rat studies are available, while we demonstrated above that dog is more sensitive than rats and mice

(4): = systemic NOAEL /Assessment factor for extrapolation to chronic exposure /Allometric scaling factor /Remaining species differences (simplified calculation to allow simple comparison of the 3 studies)

(5): lower-bound values as no adverse effect was identified in this clinical trial

(6): lower assessment factor used as the default value of 10 is for extrapolation of animal to human, and the clinical trial was in women with premenstrual tension = a sensitive, ill population

bold: final key study to derive human long-term DNEL values

This table demonstrates that the most relevant starting point to derive the human long-term systemic DNEL is the dog 14-week study (Lankas, 1981). Rats lead to a higher value which is thus not a worst-case. Since human clinical trials did not identify adverse effects, they only allow to derive lower-bound DNELs which are only tabulated to ensure that dog-derived DNELs are not overconservative. The human-derived DNEL being lower than the dog-derived DNEL, this confirms that the latter is not overconservative. Remarkably, rat-, dog- and human-derived DNELs are very consistent as they only differ by a factor of <4, illustrating once again the very high quality of the AMP toxicological database.

B) OTHER ROUTES

I. Dermal route (Carney 2006):

The available dermal study in rats did not include liver histopathology so it cannot be used to derive a dermal systemic DNEL.

AMP was adjusted to pH 9.5 in this study, so it cannot be used to derive a dermal local DNEL to protect against alkaline effects.

II. Inhalation route (Sullivan 2017):

Nose-only exposure of rats to AMP aerosol (10% AMP solution in water) for 6 hours/day for 5 days at up to 2000 mg AMP/m3 resulted in no early deaths. Three target organs were identified:

- Significant local irritation/corrosion on skin (scabs, crusts, epidermal hyperplasia, mixed cell infiltrates, necrosis and ulceration);

- Significant local irritation/corrosion in nasal cavities (atrophy of goblet cells and olfactory epithelium, epithelium hyperplasia, mixed cell infiltrates, squamous metaplasia of respiratory epithelium, ulceration of turbinates);

- Minimal systemic toxicity in liver (increased aspartate aminotransferase, decreased albumin, higher liver weight, hepatocyte vacuolation).

The LOAEC was 700 mg/m3 in both sexes. Considering 4-5% weight loss over only 5 days, the MTD ranged >=700 to <1400 mg/m3 in both sexes. At the tested 10% concentration, pH of industrial-grade AMP should be 12.3. This shows that the most sensitive general toxic effect upon inhalation of high airborne AMP concentrations is not liver toxicity but pH-related corrosion of respiratory tract and skin.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

1) The National Library of Medicines database was used to retrieve a list of Over The Counter (OTC) US drugs which contain pamabrom
2) Each drug presentation sheet was used to note down drug composition, posology, adverse effects, warnings and information when to stop treatment.

GLP compliance:

Remarks:

GLP not applicable to bibliographic search in a public database

Limit test:

Species:

other: human

Sex:

female

Route of administration:

oral: capsule

Duration of treatment / exposure:

repeated treatment cycles of up to 3 or 10 days, or longer if prescribed by a doctor

Frequency of treatment:

daily

Key result

Dose descriptor:

NOAEL

Effect level:

>= 0.73 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP fraction (in pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

based on posology of 20 OTC drugs

Critical effects observed:

In the US, pamabrom is used since early 1950's so it has a track-record of >70 years of safe use in an ill population. Based on the National Library of Medicines database, at least the 20 below US OTC drugs currently contain pamabrom:

Drug name	Indications/claims	Pamabrom content (AMP content)	Maximum daily posology	Maximum mg AMP/kg bw/day (assuming 70 kg)	Maximum duration
BACKAID MAX	For the temporary relief of: minor aches, pains and related discomforts due to muscle strain, spasms or overexertion including those affecting the back, legs and joints; pressure-caused discomforts due to periodic excess water retention	25 mg/caplet (6.4 mg/caplet)	6 caplets	0.55	10 days for pain 3 days for fever longer if prescribed
DIUREX	For the relief of following, associated with premenstrual and menstrual periods: temporary water weight gain, bloating, swelling, full feeling.	50 mg/capsule (12.8 mg/caplet)	4 capsules	0.73	10 days longer if prescribed
GREEN GUARD CRAMP RELIEF MENSTRUAL RELIEF MEDI FIRST PMS RELIEF MEDI FIRST PLUS PMS RELIEF MENSTRUAL PAIN RELIEF MAXIMUM STRENGTH MENSTRUAL PAIN RELIEF MAXIMUM STRENGTH / MULTI SYMPTOM MENSTRUAL RELIEF MAXIMUM STRENGTH MENSTRUAL RELIEF MAXIMUM STRENGTH MULTI SYMPTOM PMS RELIEF MAXIMUM STRENGTH THOMPSON CRAMP RELIEF UNISHIELD PMS RELIEF	For the temporary relief of these symptoms associated with menstrual periods: headache, bloating, cramps, backache, muscular aches, irritability, water-weight gain	25 mg/caplet (6.4 mg/caplet)	6 caplets	0.55	10 days for pain 3 days for fever longer if prescribed
PAMPRIN MULTI-SYMPTOM MAXIMUM STRENGTH PREMSYN PMS PREMENSTRUAL PAIN RELIEF	For the temporary relief of these symptoms associated with menstrual periods: cramps, headache, bloating, backache, water-weight gain, muscular aches, irritability	25 mg/caplet (6.4 mg/caplet)	8 caplets	0.73	10 days for pain 3 days for fever longer if prescribed
CRANE SAFETY CRAMP GREEN GUARD PMS RELIEF	Temporarily relieves headache, bloating, cramps, backache, water-weight gain, muscular aches and pains associated with the menstrual period	25 mg/caplet (6.4 mg/caplet)	8 caplets	0.73	not indicated
UNISHIELD CRAMP	Temporarily relieves headache, bloating, cramps, backache, water-weight gain, muscular aches and pains associated with the menstrual period	25 mg/caplet (6.4 mg/caplet)	8 caplets	0.73	10 days for pain 3 days for fever longer if prescribed
MEDI FIRST CRAMP MEDI FIRST PLUS CRAMP	For the temporary relief of minor aches and pains associated with headache, backaches, menstrual cramps. Temporarily relieves water-weight gain, bloating, swelling and full feeling associated with the premenstrual and menstrual periods	25 mg/caplet (6.4 mg/caplet)	8 caplets	0.73	not indicated

All contain the same warnings: "If pregnant or breast-feeding, ask a health professional before use."

This weight-of-evidence demonstrates that AMP is devoid of adverse effects to human health when ingested at 0.73 mg AMP/kg/day for up to 10-day treatment cycles, repeated over periods.

Conclusions:

In a target population consisting mostly (19 of 20 OTC drugs) of women with menstrual periods, AMP has a human oral NOAEL of at least 0.73 mg AMP/kg bw/day for up to 10-day treatment cycles, which can be repeated at each menstrual period.

Executive summary:

Based on the National Library of Medicines database, at least 20 US OTC drugs currently contain pamabrom, 19 of which specifically target women with menstrual periods. Based on contents and posology for each drug, AMP has a human NOAEL of at least 0.73 mg AMP/kg bw/day for up to 10-day treatment cycles, repeated over periods. This weight-of-evidence demonstrates that AMP is devoid of adverse effects to human health at this dose-level.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

repeat-dose toxicity data generated in a reproductive toxicity study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 443

Principles of method if other than guideline:

repeat-dose toxicity data generated in a reproductive toxicity study. For further details see the cross-referenced study summary.

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

99.2% pure: high-purity-grade

Based on molecular weights of this equimolar salt, AMP-HCl (CAS 3207-12-3, MW = 125.60) contains 71.0% AMP (CAS 124-68-5, MW = 89.14). Therefore AMP-HCl doses can be converted into AMP doses using a correction factor of x0.71.

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
For administration to Group 1 control animals, an appropriate amount of PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was weighed out weekly and placed in a labeled bag.
For administration to Group 2, 3 and 4 animals, an appropriate amount of the test substance for each group was added to PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 on a weight/weight basis, and mixed to form a premix. The remainder of rodent feed to achieve the desired concentration added to the pre-mix, after which the diet was blended to achieve a total batch of homogeneous diet at the appropriate concentration/group. The test diets were prepared approximately weekly, or as needed, and stored at room temperature.
ADAPTATIONS DURING GESTATION AND LACTATION
During gestation and lactation, concentration of the test substance in the diet were adjusted based on historical control data for body weights and food consumption data for gestating and lactating females in order to compensate for the higher caloric demand on maternal animals during these periods.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
Analyses were performed by an ultra-high performance liquid chromatography method using a validated analytical procedure. Analysis to demonstrate the stability and homogeneity of test diet admixes between 50 and 15,000 ppm for at least 4 and 10 days under room temperature and refrigerated (target of 5°C) conditions has been previously established by Charles River Ashland.
Homogeneity and stability of the test substance in dietary preparations prepared as low as 50 ppm were established prior to administration to study animals by Charles River Ashland.
SAMPLING
Dose formulation samples were taken weekly to monthly for the analysis of concentration (all groups). Samples for homogeneity analysis (groups 2-4) were taken from the first prepared batch only. Due to the adjustment of concentrations of the test substance in the diet beginning on PND 21, homogeneity and stability of the test substance in dietary preparations prepared as low as 50 ppm were established prior to administration of this concentration to study animals by Charles River Ashland.
ACCEPTANCE CRITERIA
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was within 20% or less at a concentration that is within the acceptable limits (80% to 120% of the target concentrations). After acceptance of the analytical results, backup samples were discarded.

Duration of treatment / exposure:

F0 FEMALES: F0 females were administered the test substance continuously in the diet for a minimum of 70 consecutive days (10 weeks) prior to mating, during mating (max 2 weeks), gestation and lactation (~3 weeks each) and until scheduled necropsy on study week 18. Total treatment duration: 18 weeks.

F0 MALES: F0 males were administered the test substance continuously in the diet over the same period as females, until scheduled necropsy on study week 18. Total treatment duration: 18 weeks.

COHORT 1A and 1B: The offspring selected for the F1 generation was indirectly exposed via F0 females, in utero and then potentially via lactation (~3 weeks each). Then they were administered test substance in the diet from weaning until fasting for scheduled necropsy on PND 91 [Cohort 1A] or 98 [Cohort 1B]). Total treatment duration: 6 weeks indirect exposure followed by 13-14 weeks direct exposure.

Frequency of treatment:

continuously through diet

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

achieved dose-levels in F0 (mg/kg/d):
50-51 in males until sacrifice and in females until end of mating
42 in females during gestation
45 in females during lactation
51-52 in M/F during F1 generation

containing 35 mg/kg bw/day AMP

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

achieved dose-levels in F0 (mg/kg/d):
100-103 in males until sacrifice and in females until end of mating
80 in females during gestation
91 in females during lactation
103 in M/F during F1 generation

containing 71 mg/kg bw/day AMP

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

achieved dose-levels in F0 (mg/kg/d):
197-203 in males until sacrifice and in females until end of mating
164 in females during gestation
181 in females during lactation
204 in M/F during F1 generation

containing 142 mg/kg bw/day AMP

No. of animals per sex per dose:

25 in F0 groups 1, 2, 3
30 in F0 group 4 (200 mg/kg/day)
21-22 (M & F) in F1 cohort 1A groups 1, 2, 3
26 M + 24 F in F1 cohort 1A group 4 (200 mg/kg/day)
19-22 (M & F) in F1 cohort 1B groups 1, 2, 3
25 M + 23 F in F1 cohort 1B group 4 (200 mg/kg/day)

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study
- General health/mortality and moribundity
DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily throughout the study
BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study and prior to necropsy Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 14, and 21. Body weights were collected prior to and after fasting.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was quantitatively measured weekly throughout the study, except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 14, and 21. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The mean amounts of test substance consumed (mg/kg bw/day) by each sex per dose group were calculated from the mean food consumed (g/kg bw/day) and the appropriate target concentration of test substance in the food (mg/kg).
THYROID HORMONE ANALYSIS
Animals were fasted overnight prior to blood collection. Blood samples for thyroid hormone analyses (Thyroxine (Total T4) and Thyroid Stimulating Hormone (TSH)) were collected (prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) from a jugular vein into tubes without anticoagulants. Samples were collected from 10 animals/sex/group in week 18.
CLINICAL PATHOLOGY
Animals were fasted overnight prior to blood collection. Urine was collected overnight using metabolism cages. Blood samples for hematology and serum chemistry were collected from a jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants. Samples were collected from 10 animals/sex/group in week 18. Hematology, coagulation, serum chemistry and urinalysis parameters were in line with OECD TG 443 guidance.

Sacrifice and pathology:

SACRIFICE
All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation in week 18
GROSS NECROPSY
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of implantation sites and former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
HISTOPATHOLOGY / ORGAN WEIGHTS
ORGAN WEIGHT: Organs of F0 animals were collected in line with OECD TG 443. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
HISTOPATHOLOGY
Tissues collected from all animals in the control and high-dose groups and from all animals euthanized in extremis, as well as gross lesions from all animals in all groups, and reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were preserved, processed and evaluated according to OECD TG 443. Processing of the testes, epidididymes, and ovaries were performed as noted below.
Testis and epididymis: Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Ovary: Five (5) sections were taken approximately 100 μm apart from the inner third of each ovary of F0 females suspected of reduced fertility. In addition, a single section was taken from remaining F0 females for a qualitative bilateral evaluation of each ovary. For females euthanized in extremis, a single section from each ovary was qualitatively evaluated. For any F0 female suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles. Uterine and ovarian histopathology were considered in light of the terminal estrous stage.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis. For males that survived to the scheduled necropsy, microscopic evaluation
included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 50 mg/kg bw/day group was euthanized in extremis on Lactation Day 8 due to poor clinical condition, including clinical observations of hunched posture, a thin and pale body, red material around the nose, and pale and cool extremities noted up to 2 days prior to euthanasia. Uterine adhesions, and one mummified fetus and 2 severely autolyzed fetuses in utero were observed grossly. Histologically, there was evidence of a uterine infection (severe inflammation with intralesional bacteria) and secondary sepsis (inflammation on the surface of multiple abdominal and thoracic organs with bacteria, decreased lymphoid cellularity in lymphoid organs, increased macrophages in the spleen, and increased myeloid cellularity in the bone marrow). Therefore, the cause of death was determined to be sepsis, secondary to the retained fetuses. All other F0 animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were noted at all determinations, except for the below.

Lower (statistically significant) mean body weight gains were noted in the 50, 100, and 200 mg/kg bw/day groups when the Lactation Days 1-21 cumulative interval was evaluated compared to the control group, albeit not in a clear dose-related manner. However, mean absolute body weights in these groups were unaffected throughout lactation; therefore, these differences were not considered test substance related.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were noted at all determinations, except for the below.

Lower (statistically significantly) mean food efficiency was noted in the 50, 100, and 200 mg/kg bw/day groups during the Lactation Days 1-21 cumulative interval compared to the control group, albeit not in a clear dose-related manner (see attached table). However, mean absolute body weights in these groups were unaffected throughout lactation; therefore, these differences were not considered test substance related.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance triggered organ weight changes in kidneys and liver of F0 generation males:

KIDNEY: There was a dose-related increase in kidney weights as absolute values and percent body weight at ≥ 50 mg/kg bw/day. Differences were statistically significant at ≥ 100 mg/kg bw/day. There were no histological correlates.

LIVER: There was a dose-related increase in all three liver weight parameters at ≥ 50 mg/kg bw/day with statistical significance at 200 mg/kg bw/day. The increased liver weights correlated histologically with hepatocellular vacuolation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related gross findings were noted, including for the number of implantation sites.

A statistically significant higher number of unaccounted for sites was noted in the 200 mg/kg/day group compared to the control group (2.7 vs. 1.0). This difference was primarily attributed to a single female in this group with 13 unaccounted-for sites. In addition, the number of unaccounted-for sites in this group was within the Charles River Ashland historical control data range, and therefore was not considered test substance-related.

F0 generation females with suspected reduced fertility (see "Reproductive performance") had quantitative counts of primordial/small growing ovarian follicles performed. Primordial/small growing ovarian follicular counts of these animals were similar to individual values obtained from the F1 Cohort 1A control females, with the exception of one animal in the 50 mg/kg bw/day group (lower ovarian follicle counts); however, this change did not occur in a dose-related manner. Test substance administration did not cause decreased primordial/small growing ovarian follicles in these F0 animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance exposure was associated with hepatocellular vacuolation in the liver of F0 generation males at ≥ 50 mg/kg bw/day with a dose-related increase in incidence and severity. This lesion was characterized by minimal to moderate vacuolation of hepatocellular cytoplasm by clear round variably sized vacuoles. The distribution was multifocal and centrilobular to random. Hepatocellular vacuolation was considered adverse at 200 mg/kg bw/day due to the moderate severity.

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOAEL

Remarks:

general/systemic toxicity

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

general/systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 71 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Remarks:

general/systemic toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

containing 142 mg/kg bw/day AMP

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

For further details see the cross-referenced study summary. Above summaries relate to the parental generation (F0). Below tables detail liver findings in both generations.

F0 / Incidence of treatment-related Liver Effects

Sex	Male				Female
Dose (mg/kg/day)	0	50	100	200	0	50	100	200
Liver (# examined)	25	25	25	30	25	0	0	30
Vacuolization, hepatocellular, multifocal, centrilobular to random:	0	4	11	24	0	-	-	0
minimal	0	4	10	10	0	-	-	0
mild	0	0	1	7	0	-	-	0
moderate	0	0	0	7	0	-	-	0

Bold type indicates the effects judged to be adverse.

F1A / Incidence of treatment-related Liver Effects

Sex	Male				Female
Dose (mg/kg/day)	0	50	100	200	0	50	100	200
Liver (# examined)	20	22	22	20	20	0	0	20
Vacuolization, hepatocellular, multifocal, centrilobular to random:	0	1	7	8	1	-	-	0
minimal	0	1	7	4	1	-	-	0
mild	0	0	0	3	0	-	-	0
moderate	0	0	0	1	0	-	-	0

Bold type indicates the effects judged to be adverse.

Conclusions:

In a rat dietary fertility toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP's parental NOAELs were 71 and 142 mg AMP/kg bw/day in males and females, resp.

Executive summary:

In an OECD 443 study, groups of 19 to 30 CD rats/sex/dose were fed diets supplying up to 200 mg AMP-HCl/kg bw/day (containing up to 142 mg AMP/kg bw/day) over two generations. Toxicologically relevant effects were limited to liver toxicity in male rats of F0 and F1A generations:

At 142 mg AMP/kg bw/day, liver weights were 12% increased in F0 only, and minimal to moderate (adverse) hepatocellular vacuolation was seen in 24/30 F0 rats and 8/20 F1A rats;

At 71 mg AMP/kg bw/day, non-adverse hepatocellular vacuolation was seen in 11/25 F0 rats (minimal to mild) and 7/22 F1A rats (minimal);

At 35 mg AMP/kg bw/day, non-adverse minimal hepatocellular vacuolation was seen in 4/25 F0 rats and 1/22 F1A rats.

The NOAEL for parental/general toxicity was 71 and 142 mg AMP/kg bw/day in males and females, resp. and the male LOAEL was 142 mg AMP/kg bw/day.

Below conclusions were drawn post-report by the registrant:

1) The absence of irreversible liver lesions means effects do not warrant STOT RE classification.

2) At the LOAEL (male F0: 142 mg AMP/kg bw/day), diet contained 3155-4801 ppm AMP-HCl (see report p. 54), i.e. 0.224-0.341 % w/w AMP. AMP was tested neutralized as AMP-HCl, CAS No. 3207-12-3. As is, AMP is alkaline (high-purity-grade: pKa = 9.70). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3189 ln(Concentration in % w/w) + 11.401], pH of high-purity-grade AMP solutions at 0.224-0.341 % AMP w/w would range 10.9-11.1. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is instead of AMP-HCl, the study's LOAEL could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines referenced by REACH do NOT require any neutralization or pH adjustment of test items, this study represents artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity.

3) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

As low/mid doses differed by a factor of 22, the male NOAEL is a wide range. Some contaminant AMP was detected in control diets.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Principles of method if other than guideline:

The protocol was discussed with the FDA personnel prior to initiation.

GLP compliance:

Limit test:

Specific details on test material used for the study:

AMP‐95 HCl, 66.1% solution - industrial grade

Based on molecular weights of this equimolar salt, AMP-HCl (CAS 3207-12-3, MW = 125.60) contains 71.0% AMP (CAS 124-68-5, MW = 89.14). Therefore AMP-HCl doses can be converted into AMP doses using a correction factor of x0.71.

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Study was run with Beagle dogs, 4/sex/dose. They were 4.5-5 months of age at arrival to the Bio/dynamics laboratory, and 5.5-6 months of age at the initiation of the study. Weight range at the start of treatment was 7.7-9.6kg for males (mean 8.7kg), and was 6.3-9.7 for females (mean 8.1kg). The dogs were immunized previously against distemper, hepatitis, leptospirosis, and rabies by the supplier. Fecal examinations were conducted on all dogs by the performing lab during the one month acclimation period. Baseline clinical laboratory tests were also performed. They were housed in elevated metal grid cages, and fed 400g of Purina Canine Meal Diet (#5007) presented for 4.5 hours. Fresh food was presented daily. Water was provided ad libitum by an automated water system. The dogs were maintained on a 12-hour photocycle, and temperature was monitored twice daily.

Dogs were ranked by body weight and distributed into 4 groups of 4/sex so that body weights in each group were comparable. Groups were assigned to control and dose levels randomly. Each dog was assigned a unique identification number on an ear tag and on the animal's cage. In addition, each dog had an ear tatoo bearing the USDA number.

Route of administration:

oral: feed

Vehicle:

other: feed

Details on oral exposure:

The AMP was administered orally via the diet as AMP-95/HCl to 32 Beagle dogs (4/sex/dose group) at dose levels of 0, 25, 600, and 2500 ppm for a three month period. The control animals received an untreated diet.
There is no information on pH of the test item or diets.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Feed samples of each dose level were assayed for homogeneity, stability, and verification of dose concentration each week.

Duration of treatment / exposure:

96 to 99 days depending on groups: 14 weeks

Frequency of treatment:

continuous

Dose / conc.:

25 ppm

Remarks:

expressed as AMP moiety; mean achieved dose-level 0.78(males) - 0.85(females) mg AMP/kg bw/day.

Dose / conc.:

600 ppm

Remarks:

expressed as AMP moiety; mean achieved dose-level 17 mg AMP/kg bw/day.

Dose / conc.:

2 500 ppm

Remarks:

expressed as AMP moiety; mean achieved dose-level 81 mg AMP/kg bw/day.

No. of animals per sex per dose:

Control animals:

yes, plain diet

Positive control:

Observations and examinations performed and frequency:

Mortality and clinical signs observed twice a day. Detailed physical examination pretest and once a week.
Ophthalmoscopic examinations were performed pretest and at termination.
Body weights were measured pretest, weekly thereafter, and after fasting at termination.
Food consumption was measured daily and calculated on weekly basis.

Hematology, clinical chemistry, and urinalysis parameters were analyzed twice pre-test, and Month 1 and 3.
Blood was obtained via jugular venipuncture. Urine was collected in stainless steel metabolism pans attached to each animal's permanent cage. Dogs were fasted overnight prior to blood collections, and were not dosed until after samples were collected.
Hematology parameters evaluated included: hemoglobin, hematocrit, erythrocytes, platelets, clotting time, prothrombin time, and total and differential leukocytes.
Clinical Chemistry parameters included: serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, lactic acid dehydrogenase, blood urea nitrogen, fasting glucose, cholesterol, total protein, albumin, globulin, A/G ratio, total bilirubin, direct bilirubin, sodium, potassium, chloride, and calcium.
Urine was evaluated for the following: Gross appearance, specific gravity, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, and microscopic analysis.

Sacrifice and pathology:

On days 96-99, animals were necropsied and tisses were preserved for weights (brain, ovaries, pituitary, thyroid, heart, adrenals, spleen, testes, kidneys, liver) and microscopic evaluation (the above plus aorta, bone, sternum, epididymis, esophagus, eye, colon, duodenum, ileum, lungs, mesenteric lymph node, mammary gland, sciatic nerve, pancreas, parathyroid, prostate, salivary gland, skeletal muscle, skin, spinal cord, stomach, thymus, thyroid, trachea, urinary bladder, and gross lesions).

Statistics:

Statistical analyses were performed on data collected for body weight, food consumption, hematology, clinical chemistry, organ weights, and organ:body weight ratios. Mean values of all dose groups were compared to control at each time interval (where appropriate). Statistically significant differences from the controls are indicated.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High-dose males and females had up to 10-11% and 7-10%, resp., lower mean body weight than controls from weeks 11 and 3, resp., to study end.
Cumulative body weight gain from pre-test to terminal body weight, was 45% and 24% lower in high-dose males and females, resp., when compared with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The only toxicologically relevant change was 16% lower hemoglobin concentration at month 3 in high-dose females vs. controls, slightly below the normal physiological range and statistically significant (p<0.05).

There were no statistically-significant differences between the control and treated animals at Month 1 testing. At Month 3, significantly reduced prothrombin time was observed in the top two dose levels for males (p<0.01). However this was not toxicologically relevant since the mean value was the same or higher (6.5 or 6.6 s) as in control males at pre-test 1 and month 1 (6.5 s).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 1 and 3 months, high-dose males and females had higher serum activities of liver enzymes (glutamic oxaloacetic transaminase 2-4 fold, glutamic pyruvic transaminase 12-28 fold, alkaline phosphatase 5-6 fold) and lower mean cholesterol levels.
The report also mentions lower bilirubin levels but this is not confirmed by raw data.

The above effects observed in the high dose animals suggest a high degree of hepatocellular damage in the high dose animals with no significant changes in the low or mid dose groups. The combination of increased serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase levels, both of which are present in high concentrations in hepatocytes, are indicative of hepatocellular necrosis. The increased alkaline phosphatase and direct bilirubin levels are suggestive of cholestasis and/or bile duct epithelial necrosis. It appears, therefore, that the liver is the primary target organ, based on the clinical chemistry data, when the test substance is administered via the diet.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A trend towards increased liver and/or liver/body weight rations was observed in both the mid and high dose male and female dogs. These differences were slight but tend to support the conclusion that the liver is the principle site of toxic action. Also, mean kidney weights and kidney/body weight ratios were increased in the high dose males, and in mid and high dose females.
Based on absence of dose-relationship and increased absolute organ weights, the differences were not considered treatment-related in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two females and one male from the high dose group were found to have tan discoloration and mottling in liver upon gross postmortem evaluations.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings included vacuolization, periportal cirrhosis, and bile duct hyperplasia in the liver sections in all high-dose males and females. Lipid deposition within the cellular vacuoles was confirmed by staining. The periportal cirrhosis was characterized by hepatocellular necrosis and fibrosis. These findings were more severe in the high-dose females as compared to the males. In addition, one low dose female had liver findings similar to the above, while 2 mid-dose males exhibited minimal fatty changes in the liver (hepatocellular vacuolization).
Minimal to moderate chronic gastritis in 2 males and 3 females at high-dose. Inflammation of gastric (sub)mucosa: non-adverse local effect, at low- and mid-dose.

The above microscopic findings correlate well with the clinical chemistry data, as both indicate hepatotoxicity induced by the test material in the high dose animals. The findings noted in the liver of the one low dose female, in the absence of altered clinical chemistry data, as well as the absence of similar microscopic findings in the mid dose group, are considered to be of uncertain significance (this may represent a response to treatment in an unusually sensitive animal, or may be a spontaneous finding).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

600 ppm

Based on:

act. ingr.

Remarks:

expressed as AMP moiety; mean achieved dose-level 17 mg AMP/kg bw/day in both sexes

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

2 500 ppm

Based on:

act. ingr.

Remarks:

expressed as AMP moiety; mean achieved dose-level 81/82 mg AMP/kg bw/day in males/females

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Achieved dietary concentrations:

Nominal concentration (ppm AMP)	0	25	600	2500
Analyzed concentration: range weeks 1-14	nd-6.0	18-54	72-699	2065-3234
Analyzed concentration: mean weeks 1-14	1.7	28	565	2578

Achieved dose-levels:

Sex	Male				Female
Nominal concentration (ppm AMP)	0	25	600	2500	0	25	600	2500
Food consumption (g/kg bw/day): mean weeks 1-14	31.9	28.2	30.7	31.2	30.4	30.8	30.7	31.7
Achieved dose-level (mg/kg bw/day): range weeks 1-14*	0.00-0.20	0.45-1.33	2.29-21.5	60.7-118	0.00-0.19	0.48-1.39	2.26-23.5	64.1-111.2
Achieved dose-level (mg/kg bw/day): mean weeks 1-14*	0.05	0.78	17.2	80.8	0.05	0.85	17.3	81.8

*Calculated post-report for each week/sex/group based on food consumption for each week/sex/group and analyzed concentration in diet for each week/group. The mean is calculated from all weekly values, weeks 1 to 14.

Organ weight data:

Sex	Male				Female
Nominal concentration (ppm AMP)	0	25	600	2500	0	25	600	2500
# examined	4	4	4	4	4	4	4	4
Liver weight:	0	0	0	0	0	0	0	0
absolute (g)	286	268	307	280	244	233	273	281
relative to terminal body weight (x1000)	2.61	2.51	2.86	2.86	2.37	2.42	2.83	2.77
Kidney weight:
absolute (g)	49.3	48.8	48.5	50.9	40.0	37.4	43.2	45.7
relative to terminal body weight (x100)	4.52	4.58	4.58	5.23	3.92	3.89	4.52	4.85

Bold type indicates treatment-related effects (absolute and relative, dose-related)

Histopathology data:

Sex	Male				Female
Nominal concentration (ppm AMP)	0	25	600	2500	0	25	600	2500
# examined	4	4	4	4	4	4	4	4
Liver
hepatocellular vacuolation - grade 1			1	1
hepatocellular vacuolation - grade 3			1	1				4
hepatocellular vacuolation - grade 4				1
congestion - grade 1				1		(1)
congestion - grade 2								1
congestion - grade 3				1				1
single cell necrosis - grade 1				3				3
single cell necrosis - grade 3						(1)
periportal fibrosis - grade 3				1		(1)		2
Kupffer cell proliferation - grade 1							2
Kupffer cell proliferation - grade 2				3
unspecific reactive hepatitis - grade 2								1
unspecific reactive hepatitis - grade 3								1
bile duct hyperplasia - grade 1				1
bile duct hyperplasia - grade 2				1
bile duct hyperplasia - grade 3						(1)
positive at Oil Red-O stain - grade 1				3				1
positive at Oil Red-O stain - grade 2				1				3
Stomach (sub)mucosa
lymphoid cell accumulation - grade 1		1		1		2
lymphoid cell accumulation - grade 2			2
lymphoid cell accumulation - grade 3		3				2
chronic inflammation - grade 1				2				1
chronic inflammation - grade 2								1
chronic inflammation - grade 3								1

Bold type indicates those effects judged to be adverse. Red indicates those judged to be irreversible.

grades: 1/minimal-very slight; 2/slight; 3/moderate; 4/moderately severe; 5/severe

(): observed in the same single female at 25 ppm, absent in 4 females at 600 ppm (0.85 -> 17.3 mg/kg bw/day i.e. 20-fold higher dose-level) = not toxicologically relevant, assumed to be a random occurence

Conclusions:

In a dog dietary 14-week toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP had a systemic NOAEL of 17 mg AMP/kg bw/day in both sexes. In addition, local toxicity (gastric inflammation) was noted at all dose-levels.

Executive summary:

In a 14-week oral repeated-dose study similar to OECD 409, beagle dogs (4/sex/dose) were fed 25, 600 or 2500 ppm AMP, provided as AMP-95 HCl, in the diet. Following effects were noted:

At 2500 ppm AMP: 45% (males) and 24% (females) lower body weight gain over the whole study. 16% lower hemoglobin concentration at month 3 in females. Higher serum activities of liver enzymes in both sexes (glutamic oxaloacetic transaminase 2-4 fold, glutamic pyruvic transaminase 12-28 fold, alkaline phosphatase 5-6 fold) and lower mean cholesterol levels in both sexes. Increased liver and kidney weights in females, non adverse per se. Tan discoloration and mottling in liver in both sexes. Multiple liver lesions including vacuolization, periportal cirrhosis (lipid deposition, hepatocellular necrosis and fibrosis) and bile duct hyperplasia in all animals. Minimal to moderate chronic gastritis in both sexes: local effect.

At 600 ppm AMP: Increased liver and kidney weight in females, not adverse per se. Non-adverse (minimal to moderate and reversible in nature) liver lesions in 2 males and 2 females. Inflammation of gastric (sub)mucosa: local effect.

At 25 ppm AMP: Inflammation of gastric (sub)mucosa: local effect.

Below conclusions were drawn post-report by the registrant:

1) Data on dietary analyses, body weight and food consumption allowed to calculate mean achieved dose-levels as 0.78 (males)/0.85 (females) mg AMP/kg bw/day at 25 ppm, 17 mg AMP/kg bw/day at 600 ppm and 81 mg AMP/kg bw/day at 2500 ppm.

2) Based on results it is proposed to derive a systemic NOAEL of 17 mg AMP/kg bw/day in both sexes (600 ppm). The gastric inflammation being a local effect, it is ignored in the systemic NOAEL.

3) At the LOAEL of 2500 ppm, achieved AMP concentrations in diet ranged 2024-3234 ppm (see report Appendix N-12), i.e. 0.202-0.323 % AMP w/w. AMP was tested neutralized as AMP-HCl, CAS No. 3207-12-3. As is, AMP is alkaline (industrial-grade: pKa = 9.74). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3309 ln(Concentration in % w/w) + 11.548], pH of industrial-grade AMP solutions at 0.202-0.323 % AMP w/w would range 11.0-11.2. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is instead of AMP-HCl, the study's LOAEL could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines referenced by REACH do NOT require any neutralization or pH adjustment of test items, this study represents artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity.

4) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

5) The irreversible liver toxicity (cirrhosis) noted at the high-dose of 81 mg AMP/kg bw/day (2500 ppm AMP, tested as AMP-HCl) does not warrant STOT RE classification for AMP, from scientific and regulatory points of view:

2500 ppm cannot been reached with non-neutralized AMP due to excess pH as explained in previous paragraph;

effects being observed with AMP-HCl (CAS N° 3207-12-3) and not AMP (CAS N° 124-68-5), they would at most trigger classification of AMP-HCl but not AMP;

effects occured close to 100 mg/kg bw/day after 14-week exposure, and the CLP classification guidance value is <100 mg/kg bw/day after 13-week exposure only;

the CLP classification guidance refers to rat studies, and nowhere does the CLP Regulation mention dog studies;

no indications of irreversible liver toxicity were reported in any of the AMP studies in rats (8 studies), mice, rabbits and monkeys (1 study each);

no liver effects were reported for pamabrom (containing AMP) in 5 human clinical trials, notably covering sensitive populations (ill or pregnant);

no liver effects/warnings are associated with pamabrom for any of the 20 OTC drugs containing it, pamabrom-based drugs being used since 70 years.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

cross-reference to human clinical trials under IUCLID section 7.10.3

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1953 to 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

see details under each of the Cross-referenced summaries

GLP compliance:

Limit test:

Specific details on test material used for the study:

see details under each of the Cross-referenced summaries

Species:

other: humans

Details on test animals or test system and environmental conditions:

see details under each of the Cross-referenced summaries

Route of administration:

oral: capsule

Duration of treatment / exposure:

NOAELs cover 3 days to 17 weeks depending on clinical trial (+ one Klimisch-4 trial summary where treatment duration was not indicated).
see details under each of the Cross-referenced summaries

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Study 1):
- Only 2/18 patients showed effects likely attributable to pamabrom since they cleared after treatment cessation: Patient n° 14 (600 mg x 67 days then 900 mg x 70 days) developed mild diarrhea after 120 days of treatment, Patient n° 11 (600 mg x 24 days) developed maculopapular rash over 4th treatment week.
- 8/18 patients showed no effects of pamabrom treatment (patients N° 4,7,8,12,13,15,16,18 treated for 13-70 days at 600 to 900 mg/day), and we can also consider patient n°14 until diarrhea appeared on day 120 (600 mg x 67 days + 900 mg x 52 days before diarrhea).
- 8/18 patients showed adverse reactions which cannot be interpreted concerning relationship
with Pamabrom treatment because of the severe cardiac failure, co-treatment with highly toxic
mercurial diuretics in most patients, and often missing information about recovery upon cessation of pamabrom treatment.

Study 2): No toxic effects noted.

Study 3): The only effects were therapeutic (benefic): variable "degree of improvement" of the premenstrual tension condition (less water retention, nervous symptoms, acne, headache, breast engorgement, gastrointestinal symptoms, pelvic pain). No toxic effects.

Study 4): Only 6/189 2 (3%) patients reported minor "adverse" effects, among which only 3/189 were not similar to symptoms already present before treatment in this ill population (primary dysmenorrhea): somnolence (one case), dizziness (one case), increased thirst (one case).

Study 5): The only effects were therapeutic (benefic): edema reduction or persistence. No toxic effects.

Study 6): A few patients complained of nausea, vomiting, etc. due to pregnancy. The only effects were therapeutic (benefic): edema reduction or persistence. No toxic effects.

Mortality:

no mortality observed

Description (incidence):

All 6 studies: no deaths reported

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Studies 1), 3), 5) and 6): The only effects were therapeutic (benefic): weight reduction /less weight gain /less "water retention" in some patients.

Studies 2) and 4) do not include any information

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not specified

Description (incidence and severity):

Study 2) investigated but no results provided

Not assessed in other studies

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

study 2): no adverse effect

Not assessed in other studies

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

study 2): no adverse effect

Not assessed in other studies

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Study 1): 7/18 patients showed no adverse effects at medical examination; other effects observed cannot be interpreted due to illness and co-treatment with toxic mercurial drugs.

Study 2): no adverse effect

Studies 3) and 4) did not include urinalysis

Study 5): The only effects were therapeutic (benefic): changes in electrolytes.

Study 6): The only effects were therapeutic (benefic): albuminuria was reduced or eliminated.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Reproduction endpoints:

Studies 1) and 2) No adverse clinical effects related to reproduction were reported.

Study 3): The only effects were therapeutic (benefic): normalisation of symptoms of premenstrual tension (menstrual cycle length, amount of flow).

Study 4): The only effects were therapeutic (benefic): normalisation of symptoms of menstrual pain.

Study 5): No adverse effects on gravidity. gestation, changes in fetal movement.

Study 6): No adverse effects reported during pregnancy.

Dose descriptor:

NOAEL

Effect level:

>= 2.2 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

male/female

Basis for effect level:

clinical signs

Remarks on result:

other: Study 1), NOAEL covers 3 to 17 weeks depending on patient

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2.9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Study 2), 28 days

Dose descriptor:

NOAEL

Effect level:

>= 1.5 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Study 3), variable treatment regime, mean 2 weeks

Dose descriptor:

NOAEL

Effect level:

>= 0.32 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Study 4), 3-day treatment in 189 patients, only 3 reported minor effects

Dose descriptor:

NOAEL

Effect level:

>= 5.9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Study 5), 5-7 days, pregnant women

Dose descriptor:

NOAEL

Effect level:

>= 2.9 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Study 5), total of 2-week treatment in 9 patients who failed to loose weight, pregnant women

Dose descriptor:

NOAEL

Effect level:

>= 2.2 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Remarks:

AMP (supplied as Pamabrom)

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Study 6), unknown treatment duration and schedule, pregnant women

Critical effects observed:

see details under each of the Cross-referenced summaries

Conclusions:

see details under each of the Cross-referenced summaries

Executive summary:

Six clinical studies are available on pamabrom. NOAELs are expressed in AMP based on pamabrom posology and AMP content.

They inform on absence of repeat-dose adverse effects, in all cases the below-listed NOAELs were the maximum tested dose:

1) Trial in severe cardiac failure patients (Doherty et al, 1953): Patients (n=19) were given pamabrom at 300 to 900 mg/day for 5 to 137 days. This study included clinical signs, edema, degree of cardiac failure, subjective improvement. Only 2/18 patients reported adverse effects potentially related to pamabrom upon longer treatment: 1/18 case of maculopapular rash (4th week of treatment) and 1/18 case of diarrhea (day 120 of treatment). A third patient stopped treatment after 2 weeks due to therapeutic success. The NOAEL was >= 2.2 mg AMP/kg bw/day x 2 to 17 weeks depending on patient.

2) Trial in women with premenstrual tension - Preliminary study (McGavack et al, 1956): oral treatment (n=9) up to 800 mg pamabrom for 4 weeks (28-days) in women with menstrual cycles. This study included general examination for toxic effects. No adverse effect. The NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks.

3) Trial in women with premenstrual tension - Main study (McGavack et al, 1956): oral treatment (n=43) with 100 to 400 mg pamabrom per day, provided in 1 to 14 successive treatment cycles (mean 2-3 treatment cycles per group) of 3-10 days before start of menstruation, mean cumulated treatment duration 2 weeks. This study included general examination and monitoring of degree of improvement of premenstrual tension (less water retention, nervous symptoms, acne, headache, breast engorgement, gastrointestinal symptoms, pelvic pain, menstrual cycle length, amount of flow). No adverse effect: the only effects were therapeutic (benefic): normalisation of symptoms of premenstrual tension (menstrual cycle length, amount of flow). The NOAEL was >= 1.5 mg AMP/kg bw/day x 2 weeks (mean: variable treatment regime).

4) Trial in women with primary dysmenorrhea (Ortiz et al, 2016): n=189 women with mean age 21 years were given 75 mg pamabrom daily for 3 days, in combination with various other drugs. The study involved monitoring of adverse clinical effects and multiple efficacy endpoints (menstrual pain reduction, symptoms of dysmenorrhea, overall clinical response). The only effects were minor effects in 3/189 patients (somnolence, dizziness and increased thirst, 1 case each) and normalisation of symptoms of menstrual pain. The NOAEL was >= 0.32 mg AMP/kg bw/day x 3 days.

5) Trial in pregnant women (Patterson, 1958): >22 week pregnant women with edema classified as mild pre-eclampsia (n=38) were given orally 800 mg of pamabrom daily for 5-7 days. Then, (n=9) patients who failed to loose weight were again given orally 1600 mg of pamabrom daily for 5-7 days. This study included monitoring of edema before and after treatment, nausea, vomiting, edema, changes in vision, headache, miscellaneous side effects, blood pressure, daily intake and output of fluids, gravidity, gestation, fetal movement. No adverse effects. The NOAEL was >= 5.9 mg AMP/kg bw/day x 1 week (if considering only the second treatment cycle), and>= 2.9 mg AMP/kg bw/day x 2 weeks (if considering both treatment cycles in patients treated twice).

6) Trial in pregnant women (James et al, 1957): In 180 pregnant women with edema treated with pamabrom for unknown duration (Klimisch 4 study), no adverse effects were reported up to 12 tablets/day and an average of 8-10 tablets/day. The max. NOAEL was >= 2.2 mg AMP/kg bw/day and in most patients it was >=1.6 mg AMP/kg bw/day.

Considering all data, the most robust human NOAEL for repeat-dose toxicity comes from study 2) (preliminary study by McGavack et al, 1956) since this is the only study monitoring hematology and blood biochemistry. The NOAEL was >= 2.9 mg AMP/kg bw/day x 4 weeks.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 17 mg/kg bw/day
Study duration:: subchronic
Species:: dog
Quality of whole database:: very high
System:: hepatobiliary
Organ:: liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

- Principle of test: The objective of this study was to determine the toxicity via inhalation exposure of AMP administered by nose-only inhalation during/ after five consecutive daily exposures
- Short description of test conditions: The rats were exposed to three dose levels of test article (700, 1400, and 2000 mg/m3) for six hours per day.
- Parameters analysed / observed:Experimental endpoints consisted of moribundity/mortality and cage-side clinical observations (pre- and post-exposure); body weights; food consumption; clinical pathology parameters (clinical chemistry and hematology); organ weights; and necropsy and histopathological evaluations (lungs, liver, kidney, spleen, adrenals, heart, nasal turbinates, and gross lesions).

GLP compliance:

Limit test:

Specific details on test material used for the study:

AMP-REGULAR™ - industrial grade

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty-two (22) male and 22 female Sprague–Dawley derived rats [Crl:CD®(CD)BR] were obtained from Wilmington, MA-based Charles River Laboratories’ facility in Stone Ridge, NY, and were received at IITRI on October 26, 2016. One day after receipt, weights of animals ranged from 183 to 219 g for males and 152 to 182 g for females. The rats were approximately seven weeks old at arrival (date of birth of September 5, 2016) and approximately eight weeks old at the initiation of dosing.
During the one-week quarantine period, the rats were observed daily for mortality or evidence of moribundity. Before being released from quarantine, the animals were carefully examined (hand-held physical examination) to ensure their health and suitability as test subjects. The rats were released from quarantine on November 2, 2016. Group Assignment and Identification: Animals were randomized with a computerized body weight stratification procedure that produced similar group mean body weight values (not exceeding ±20%) on November 1, 2016, using ToxData®. The animals were randomized into four groups. Each animal selected for the study received a permanent identification number by permanent marker on the tail at the time of randomization. Extra animals not selected for the study were used as training animals. All cages were identified by project number, study number, animal number, dose group, and sex. Cage cards were color-coded by study group.
Food and Water: The rats were provided with Certified Global 18% Protein Rodent Diet [2018C; Teklad Laboratory Animal Diets, Envigo; Madison, WI]. City of Chicago water was supplied by means of an in-cage automatic watering system. No known contaminants that would have interfered with the outcome of the study were present in the food or water of the animals. Reports for the food and water analyses are maintained with facility records. The animals did not have access to food or water during inhalation exposures or to food during the scheduled fasting periods.

Housing and Environment: During quarantine and non-exposure periods of the treatment phase, rats were double-housed in polycarbonate “shoe-box” cages (10 ½” × 19” × 8”) lined with autoclaved absorbent hardwood chip bedding. The cages were equipped with automatic feeding and watering systems. Racks and cages were sanitized following group assignment.
Temperature and relative humidity (%RH) values were recorded once daily during quarantine and twice daily during the treatment period of the study. Recorded values ranged from 21°C to 22°C and from 35% to 51% RH. Fluorescent lighting in the animal room was provided on a cycle of 12 hours of light followed by 12 hours of darkness.
Restraint and Acclimation to Restraint: During the inhalation exposures, the animals were restrained in nose-only holding tubes (CH Technologies, USA; Westwood, NJ). Following confirmation of the correct animal number, each tube was placed in a pre-designated port of the inhalation exposure chamber. Animal placement for each exposure is documented in the study records. Processes for animal tube loading and unloading and tube insertion and removal from the chamber manifold were performed according to laboratory standard operating procedures that are designed to minimize stress to the rats. The rats were observed frequently while restrained to ensure that they remained in the tubes and were not in danger of injury or death. At the end of each exposure, when the chamber was purged of the test substance, the tubes with the animals were removed. The rats were removed from the tubes, observed, and returned to their home cages. The holding tubes were sanitized after each use.
To condition the animals to placement and restraint in nose-only holding tubes and to reduce stress during the exposure phase, the animals were placed in the holding tubes for varying lengths of time on weekdays (but not on any intervening weekend) prior to the start of exposure to the test substance according to the following schedule: 1.5 hours on Day -5; 3 hours on Day -2; and 4.5 hours on Day -1 (October 28 and 31 and November 1, 2016, respectively).
Animal Welfare: The study complied with all applicable sections of the Animal Welfare Act (AWA; Title 9, Code of Federal Regulations), the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (National Institute of Health’s Office of Laboratory Animal Welfare, 2015), and the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). To the extent possible, procedures used in this study were designed to avoid or minimize discomfort, stress, and pain to the animals.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

water

Remarks:

The test article was diluted to 10% with ASTM water by weight and was used as an aerosolized aqueous solution.

Mass median aerodynamic diameter (MMAD):

>= 0.74 - <= 1.65 µm

Remarks on MMAD:

Particle Size Distribution: Mean MMAD values in test atmosphere were 1.10, 0.93, and 1.26 μm for Groups 2-4, respectively. GSD ranges in the test atmosphere were 1.67-1.95, 1.53-1.85, and 1.55-2.07 for Groups 2-4, respectively.

Details on inhalation exposure:

Test Atmosphere Concentration: Overall mean test atmosphere concentrations were 0.697 ± 0.0812, 1.614 ± 0.0986, and 2.004 ± 0.1211 mg/L for Groups 2-4, respectively.
Temperature and Humidity: Overall mean chamber temperatures were 21.8, 21.6, 21.9, and 21.3°C for Groups 1-4, respectively. Overall mean humidity levels were 14.4, 99.9, 99.9, and 99.9% for Groups 1-4, respectively; the elevated relative humidity values for Groups 2-4 were due to the aqueous nature of the dosing formulations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Impinger Analysis: One test atmosphere sample was collected per two hours of exposure (three samples per 6-hour exposure). Chamber aerosol/vapor test atmosphere concentration was monitored with a serially connected train of two impingers, each containing 20 mL of a naphthyl isothiocyanate trapping solution. Samples from each impinger were combined and brought to volume (50 mL) with isopropyl alcohol. These samples were collected at a constant flow rate equal to the port flow of the delivery tube, and the total volume of air sampled was measured with a dry-gas meter. The concentrations of the samples were quantified by chemical analysis at IITRI.
Aerosol Particle Size Distribution: Aerosol particle size distribution was determined once per day for Groups 2-4 with a quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc.; Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the mass accumulated on each collection stage of the QCM.
Aerosol Concentration: Aerosol concentration was monitored with a real-time aerosol sensor (model #pDR-1000AN; MIE Inc., Bedford, MA). The sensor was employed as a real-time indicator of short-term changes in aerosol concentration and was used in guiding laboratory personnel if concentration excursions were encountered.

Duration of treatment / exposure:

Experimental Design: The rats were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days. The first day of exposure was November 2, 2016, and the final day of exposure was November 6, 2016 (Day 5). All study animals were euthanized on November 7, 2016.
Inhalation Exposure Methods
1. Inhalation Exposure Laboratory: The study was conducted in Chambers 1-4 of Laboratory VIII. This laboratory is equipped with 64-port, flow-past-type nose-only inhalation exposure chambers (manufactured by Lab Products Inc.; Seaford, DE). A schematic diagram of the inhalation exposure system is provided in Figure 1.
Air for test atmosphere generation was of breathable quality and was filtered with a compressed air filter and a carbon absorber. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure. During exposure, the animals were held in clear plastic restraining devices (holding tubes; see Section II.C.5) attached to the chamber at the ports.
2. Test Atmosphere Generation: The test atmosphere was generated via aerosolization of the test (10% aqueous solution of AMP-REGULAR™) article using a Pari LC Plus nebulizer (Pari Respiratory Equipment, Inc.; Midlothian, VA). The test article was dispensed into the nebulizer reservoir as needed by means of a syringe. The resulting test atmosphere entered the nose-only inhalation exposure chamber. The exhaust from the exposure chamber was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotameters.

Frequency of treatment:

six hours per day for five days

Dose / conc.:

700 mg/m³ air (nominal)

Remarks:

achieved: 697 mg/m3

Dose / conc.:

1 400 mg/m³ air (nominal)

Remarks:

achieved: 1614 mg/m3

Dose / conc.:

2 000 mg/m³ air (nominal)

Remarks:

achieved: 2004 mg/m3

No. of animals per sex per dose:

Control animals:

yes, sham-exposed

Details on study design:

The rats ( 5 male and 5 female per dose group) were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days.

Observations and examinations performed and frequency:

Moribundity/Mortality Observations: Rats were observed for mortality and evidence of moribundity at least once daily prior to the initiation of dosing and at least twice daily during the treatment period. Mortality/moribundity checks were separated by a minimum of four hours, as appropriate.
Physical Examinations/Clinical Observations: Upon initiation of inhalation exposure, animals were observed regularly during exposure to correct any potential emergency conditions while in the holding tubes and to monitor for signs of toxicity (documented manually). Additionally, all surviving study animals were observed at least twice daily (before exposure and within an hour after exposure termination) for clinical signs (documented electronically in ToxData®). Observations included but were not limited to changes in the skin and fur, eyes, and mucous membranes; effects on the respiratory, circulatory, autonomic, and central nervous systems; and effects on somatomotor activity and behavior pattern. Particular attention was devoted to the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. Any animals in possibly moribund condition were identified for further monitoring and possible euthanasia.
Body Weights and Body Weight Changes: Animals were weighed one day after receipt; on the day of randomization; and on Study Days 1, 3, and 5. All study animals were fasted overnight and also weighed on Day 6 prior to scheduled necropsy.
Food Consumption: Average food consumption per cage (due to the study animals being double-housed) was recorded on the same schedule as body weights.
Clinical Pathology: Samples were collected for analysis of clinical pathology parameters from all animals prior to scheduled euthanasia on Day 6. Animals were fasted overnight prior to blood collection. Blood samples for hematology and clinical chemistry were obtained from the retro-orbital plexus under anesthesia with 70% CO2/30% O2. The following clinical pathology parameters were evaluated:
a. Hematology: Hematology blood samples were collected into tubes containing EDTA as the anticoagulant. Blood smears were prepared from fresh blood and stained with Wright–Giemsa stain for manual differential leukocyte counts but were to be evaluated only if requested by the Sponsor. The parameters listed below were evaluated using an ADVIA 120 Hematology System Analyzer (Siemens Healthcare Diagnostics; Tarrytown, NY).
Differential white blood cell count (absolute and relative)
Erythrocyte count
Hematocrit
Hemoglobin
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Mean corpuscular volume
Platelet count
Reticulocyte count (absolute and relative)
Total white blood cell count

Clinical Chemistry: Clinical chemistry blood samples were collected into tubes, allowed to clot, centrifuged to obtain serum, and assayed on the day of collection. The parameters listed below were evaluated using a Beckman Coulter AU480 Clinical System (Beckman Coulter, Inc.; Brea, CA).
Alanine aminotransferase
Creatinine
Albumin
Gamma-glutamyl transpeptidase
Albumin/globulin ratio (calculated)
Globulin (calculated)
Alkaline phosphatase
Glucose
Aspartate aminotransferase
Inorganic phosphorus
Bilirubin (total)
Lactate dehydrogenase
Blood urea nitrogen
Potassium
Calcium
Protein (total)
Chloride
Sodium
Cholesterol
Triglycerides

Sacrifice and pathology:

Postmortem Procedures
1. Necropsy, Organ Weights, Tissue Analysis, and Gross Pathology: A complete necropsy was scheduled for all animals on Day 6. Prior to necropsy, the rats were fasted overnight. Animals were euthanized by an overdose of an intraperitoneal injection of sodium pentobarbital and exsanguinated.
At scheduled necropsy, the external surface of the body; all orifices; and the cranial, thoracic, and peritoneal cavities and their contents were examined, and any lesions or abnormal conditions (gross pathologic findings) were recorded. Complete necropsies were performed in the presence/under the supervision of a pathologist.
The tissues listed in the table below were collected and fixed in 10% neutral buffered formalin with the following exceptions: The eyes (with optic nerves) were fixed in Davidson’s solution; the testes and epididymides were fixed in modified
Davidson’s solution; and the bone marrow smear was fixed in methanol. The brain, paired kidneys, liver, adrenals, testes, ovaries, and paired lungs were weighed, and organ-to-body weight ratios were calculated using the fasted body weight for each animal.
Adrenal gland (paired)
Animal Identification (tail)1
Aorta
Bone, femur
Bone, sternum
Bone marrow, femur
Bone marrow, sternum
Bone marrow smear (femur)
Brain
Cervix
Epididymis (paired)
Esophagus
Eye (paired)
Gross lesion (if any)
Harderian gland (paired)
Heart
Kidney (paired)
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Liver
Lung (paired)
Lymph node, mandibular
Lymph node, mesenteric
Mammary gland (females)
Mass (if any)
Nasal cavity and turbinates2
Nerve, optic (paired)
Nerve, sciatic
Ovary (paired)
Pancreas
Parathyroid gland (paired)3
Pituitary gland
Prostate gland
Salivary gland (paired)
Seminal vesicle (paired)
Skeletal muscle
Skin (ventral abdomen)
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord, cervical
Spinal cord, lumbar
Spinal cord, thoracic
Spleen
Stomach
Testis (paired)
Thymus
Thyroid gland (paired)
Trachea
Urinary bladder
Uterus
Vagina
Zymbal gland (paired)3
1 The tail with the identification number of each animal was collected but not processed.
2 Per the request of the Sponsor, as noted in Protocol Amendment No. 2.
3 Due to size constraints, these organs were only evaluated when present in normal sections.
Histopathology: Protocol-specified tissues required for microscopic evaluation (lungs, liver, kidney, spleen, adrenals, heart, and gross lesions) from animals in Groups 1 and 4 (Air Control and High Dose, respectively); liver and gross lesions on the skin (mostly on the nose) from animals in Groups 2 and 3 (Low and Mid Dose, respectively), as noted in Protocol Amendment No. 1; and nasal turbinates from all animals, as noted in Protocol Amendment No. 2 were trimmed, processed routinely, embedded in paraffin, and stained with hematoxylin and eosin by Charles River Laboratories, Pathology Associates, Illinois. Light microscopic evaluation was conducted by a board-certified veterinary pathologist on the protocol-specified tissues.

Statistics:

Statistical Procedures: Descriptive statistics (mean and standard deviation) were calculated, and data were analyzed for statistical significance for body weight/body weight change, clinical pathology (clinical chemistry and hematology), and organ weight/organ-to-body weight ratio data using the ToxData® system. If a data set was normally distributed and of equal variance, statistical comparisons were conducted using a one-way analysis of variance (ANOVA), with post hoc comparisons made (if necessary) using Dunnett’s test. If normality and/or equal variance failed for a data set, statistical comparisons were conducted using nonparametric Kruskal–Wallis ANOVA, with post hoc comparisons made (if necessary) using Dunn’s test. A minimum significance level of p < 0.05 was used for the statistical comparisons in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test article-related clinical observation of scabbing (head and/or forelimb) due to irritation (burning) caused by the test article:
High- and Mid-dose: all animals.
Low-dose: one rat/sex

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight loss occurred in high- and mid-dose males (-5%) and females (-4%) over 5 days.
At the low-dose, body weight gain was slightly lower in both sexes by a few grams, not reaching statistical or toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

2-fold increased monocyte count in Mid and High dose males and females were considered treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased aspartate aminotransferase (+76% to +129%) and slightly decreased albumin (-9% to -17%) in the Mid and High dose males and females were considered treatment-related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant effects on adrenal (Group 4 males); liver (Group 3 and 4 males and females); and kidney (Group 3 and 4 females) absolute and/or relative weights were recorded.

Considering histopathology data and the decreased terminal body weight, the only biologically relevant change was increased absolute liver weight:
+8% in high-dose males,
+11% and +14% in mid- and high-dose females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Skin crusts (thick hard black regions, predominantly on the nose) were observed in all mid- and high-dose animals, and in one low-dose female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High Dose: microscopic findings were noted in skin, nasal cavities and liver of 9/10 to 10/10 animals:
- skin: minimal epidermal hyperplasia, mild to moderate mixed cell infiltrates, and mild to marked necrosis and ulceration
- nasal cavities: mild to severe atrophy of goblet cells, minimal to mild atrophy of olfactory epithelium, minimal to mild epithelium hyperplasia, minimal to moderate mixed cell infiltrates, minimal to moderate squamous metaplasia of respiratory epithelium, mild to moderate ulceration of turbinates
- liver: minimal vacuolation

Mid Dose: microscopic findings were noted in skin, nasal cavities and liver of 8/10 to 10/10 animals:
- skin: minimal to marked serocellular crusts, minimal epidermal hyperplasia, mild to moderate mixed cell infiltrates, and mild to marked necrosis and ulceration
- nasal cavities: mild to marked atrophy of goblet cells, minimal to mild atrophy of olfactory epithelium, minimal to mild epithelium hyperplasia, minimal to moderate mixed cell infiltrates, minimal to moderate squamous metaplasia of respiratory epithelium, minimal to mild ulceration of turbinates
- liver: minimal to mild vacuolation

Low Dose: microscopic findings were noted in nasal cavities of 10/10 animals, and skin and liver of 1/10 animal:
- skin: moderate serocellular crusts, minimal necrosis and ulceration
- nasal cavities: minimal to moderate atrophy of goblet cells, minimal to mild atrophy of olfactory epithelium, mild epithelium hyperplasia, minimal to mild mixed cell infiltrates, minimal to mild squamous metaplasia of respiratory epithelium, minimal ulceration of turbinates
- liver: minimal vacuolation, also noted in one control

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

other: Maximum tolerated dose (MTD)

Effect level:

>= 700 - < 1 400 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

LOAEC

Effect level:

<= 700 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

dermal irritation

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: local effects (skin/respiratory tract corrosion)

Key result

Dose descriptor:

LOEC

Effect level:

1 400 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: systemic effects (liver)

Key result

Dose descriptor:

NOEC

Effect level:

700 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: systemic effects (liver)

Critical effects observed:

yes

Lowest effective dose / conc.:

700 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Target organs in order of decreasing sensitivity: respiratory tract >> skin > liver.

Effects on respiratory tract and skin are attributable to the alkaline nature of the test item (see executive summary).

Incidence of treatment-related microscopic liver effects compared to local toxicity (summarized)

Sex	Male				Female
Airborne concentration (mg/m3)	0	700	1400	2000	0	700	1400	2000
# rats examined	5	5	5	5	5	5	5	5
Liver reversible systemic toxicity (vacuolation)	0	0	3 (minimal)	5 (minimal)	1 (minimal)	1 (minimal)	5 (minimal to mild)	4 (minimal)
Skin irreversible local toxicity (necrosis and/or ulceration)	0	0	5 (mild to marked)	5 (mild to marked)	0	1 (minimal)	5 (moderate to marked)	5 (mild to marked)
Respiratory tract irreversible local toxicity (ulceration in turbinates)	0	2 (minimal)	3 (minimal to mild)	3 (mild to moderate)	0	0	2 (minimal to mild)	3 (mild to moderate)

Bold type indicates the effects judged to be treatment related.

Conclusions:

In rats exposed nose-only, 6h/day for 5 days to a 10% solution of AMP-REGULAR, the LOAEC was 700 mg/m3 in both sexes. The maximum tolerated dose ranged >=700 to <1400 mg/m3 in both sexes. Target organs in order of decreasing sensitivity were: respiratory tract >> skin > liver. Effects on respiratory tract and skin were due to the alkaline test item (estimated pH for 10% aqueous solution: 11.9).

Executive summary:

The objective of this study was to determine the toxicity via inhalation exposure of AMP administered by nose-only inhalation during five consecutive daily exposures. The following effcets were noted in rats exposed to the test aerosol (MMAD ranging 0.74-1.65 µm, 10% AMP solution in water) for six hours per day:

2000 mg/m3: skin scabbing (burns from test item) in all animals. Slight body weight loss in both sexes. Increased monocyte counts and aspartate aminotransferase and slightly decreased albumin in both sexes. 8% (males) to 14% (females) higher liver weight. Skin crusts (thick hard black regions, predominantly on the nose) in all animals. Microscopic findings in almost all animals: 1) nasal cavities: mild to severe atrophy of goblet cells, minimal to mild atrophy of olfactory epithelium, minimal to mild epithelium hyperplasia, minimal to moderate mixed cell infiltrates, minimal to moderate squamous metaplasia of respiratory epithelium, mild to moderate ulceration of turbinates. 2) skin: minimal epidermal hyperplasia, mild to moderate mixed cell infiltrates, and mild to marked necrosis and ulceration. 3) Liver: minimal vacuolation.

1400 mg/m3: skin scabbing in all animals. Slight body weight loss in both sexes. 2-fold increased monocyte counts and aspartate aminotransferase and slightly decreased albumin in both sexes. 11% (females) higher liver weight. Skin crusts in all animals. Microscopic findings in almost all animals: 1) nasal cavities (maximum severity "marked"), 2) skin (maximum severity "marked") and 3) liver (maximum severity "mild").

700 mg/m3: Skin scabbing and/or crusts in 1 rat/sex. Microscopic findings in nasal cavities in all animals (maximum severity "moderate"). In a single animal, skin lesions (maximum severity "moderate") and liver lesions (minimal vacuolation, also noted in one control).

Below conclusions were drawn post-report by the registrant:

1) The test article was a 10% solution in water and its pH was not indicated. As is, AMP is alkaline (industrial-grade: pKa = 9.74). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3309 ln(Concentration in % w/w) + 11.548], pH of industrial-grade AMP solutions at 10 % AMP w/w would be 12.3. This confirms that the observed skin and respiratory tract lesions represent corrosive effects. (local toxicity).

2) Based on corrosion to skin/respiratory tract, the local LOAEC was <=700 mg/m3 in both sexes. Based on minimal liver vacuolation (unsure whether the minimal grade also observed in one control female can even be considered adverse), the systemic NOEC and LOEC were 700 and 1400 mg/m3 in both sexes, resp. Minimal liver vacuolation being typically a reversible effect, STOT classification is not required. Considering 4-5% weight loss over only 5 days, the maximum tolerated dose ranged >=700 to <1400 mg/m3 in both sexes.

3) This study shows that the most sensitive toxic effect upon inhalation of high airborne concentrations of non-neutralised AMP is not liver toxicity (which was minimal) but pH-related local corrosion of respiratory tract and skin. Thus, liver toxicity can be avoided by keeping concentrations below corrosive ones.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEC
: 700 mg/m³
Study duration:: subacute
Experimental exposure time per week (hours/week):: 30
Species:: rat
Quality of whole database:: limited (1 study of 5 days)
System:: hepatobiliary
Organ:: liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

GLP compliance:

Limit test:

Specific details on test material used for the study:

AMP-REGULAR™ - industrial grade

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

water

Remarks:

The test article was diluted to 10% with ASTM water by weight and was used as an aerosolized aqueous solution.

Mass median aerodynamic diameter (MMAD):

>= 0.74 - <= 1.65 µm

Remarks on MMAD:

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

Frequency of treatment:

six hours per day for five days

Dose / conc.:

700 mg/m³ air (nominal)

Remarks:

achieved: 697 mg/m3

Dose / conc.:

1 400 mg/m³ air (nominal)

Remarks:

achieved: 1614 mg/m3

Dose / conc.:

2 000 mg/m³ air (nominal)

Remarks:

achieved: 2004 mg/m3

No. of animals per sex per dose:

Control animals:

yes, sham-exposed

Details on study design:

The rats ( 5 male and 5 female per dose group) were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days.

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test article-related clinical observation of scabbing (head and/or forelimb) due to irritation (burning) caused by the test article:
High- and Mid-dose: all animals.
Low-dose: one rat/sex

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

2-fold increased monocyte count in Mid and High dose males and females were considered treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased aspartate aminotransferase (+76% to +129%) and slightly decreased albumin (-9% to -17%) in the Mid and High dose males and females were considered treatment-related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Skin crusts (thick hard black regions, predominantly on the nose) were observed in all mid- and high-dose animals, and in one low-dose female.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

other: Maximum tolerated dose (MTD)

Effect level:

>= 700 - < 1 400 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

LOAEC

Effect level:

<= 700 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

dermal irritation

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: local effects (skin/respiratory tract corrosion)

Key result

Dose descriptor:

LOEC

Effect level:

1 400 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: systemic effects (liver)

Key result

Dose descriptor:

NOEC

Effect level:

700 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: systemic effects (liver)

Critical effects observed:

yes

Lowest effective dose / conc.:

700 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Target organs in order of decreasing sensitivity: respiratory tract >> skin > liver.

Effects on respiratory tract and skin are attributable to the alkaline nature of the test item (see executive summary).

Incidence of treatment-related microscopic liver effects compared to local toxicity (summarized)

Sex	Male				Female
Airborne concentration (mg/m3)	0	700	1400	2000	0	700	1400	2000
# rats examined	5	5	5	5	5	5	5	5
Liver reversible systemic toxicity (vacuolation)	0	0	3 (minimal)	5 (minimal)	1 (minimal)	1 (minimal)	5 (minimal to mild)	4 (minimal)
Skin irreversible local toxicity (necrosis and/or ulceration)	0	0	5 (mild to marked)	5 (mild to marked)	0	1 (minimal)	5 (moderate to marked)	5 (mild to marked)
Respiratory tract irreversible local toxicity (ulceration in turbinates)	0	2 (minimal)	3 (minimal to mild)	3 (mild to moderate)	0	0	2 (minimal to mild)	3 (mild to moderate)

Bold type indicates the effects judged to be treatment related.

Conclusions:

Executive summary:

2000 mg/m3: skin scabbing (burns from test item) in all animals. Slight body weight loss in both sexes. Increased monocyte counts and aspartate aminotransferase and slightly decreased albumin in both sexes. 8% (males) to 14% (females) higher liver weight. Skin crusts (thick hard black regions, predominantly on the nose) in all animals. Microscopic findings in almost all animals: 1) nasal cavities: mild to severe atrophy of goblet cells, minimal to mild atrophy of olfactory epithelium, minimal to mild epithelium hyperplasia, minimal to moderate mixed cell infiltrates, minimal to moderate squamous metaplasia of respiratory epithelium, mild to moderate ulceration of turbinates. 2) skin: minimal epidermal hyperplasia, mild to moderate mixed cell infiltrates, and mild to marked necrosis and ulceration. 3) Liver: minimal vacuolation.

1400 mg/m3: skin scabbing in all animals. Slight body weight loss in both sexes. 2-fold increased monocyte counts and aspartate aminotransferase and slightly decreased albumin in both sexes. 11% (females) higher liver weight. Skin crusts in all animals. Microscopic findings in almost all animals: 1) nasal cavities (maximum severity "marked"), 2) skin (maximum severity "marked") and 3) liver (maximum severity "mild").

700 mg/m3: Skin scabbing and/or crusts in 1 rat/sex. Microscopic findings in nasal cavities in all animals (maximum severity "moderate"). In a single animal, skin lesions (maximum severity "moderate") and liver lesions (minimal vacuolation, also noted in one control).

Below conclusions were drawn post-report by the registrant:

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: LOAEC
: 700 mg/m³
Study duration:: subacute
Species:: rat
Quality of whole database:: limited (1 study of 5 days)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available
Quality of whole database:: studies by dermal route did not investigate repeat-dose toxicity endpoints (notably liver toxicity)

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available
Quality of whole database:: studies by dermal route involved adjustment of solutions to pH 9.5 and are thus irrelevant to determine a cut-off for local effects

Mode of Action Analysis / Human Relevance Framework

Mechanistic studies (see last 5 summaries under IUCLID § 7.5.1):

Numerous mechanistic studies have been performed between 1961 and 2019 to investigate liver effects of AMP. The following 5 mechanistic studies present as IUCLID summaries can be summarized as follows:

In vitro:

Slattery 2018/2019:

In vitro, high-purity AMP and industrial-grade AMP (AMP95) both induced cytotoxicity, phospholipidosis and steatosis in a dose-related manner in rat and human hepatocytes.

The similar effects with both grades indicate that impurities do not play a significant role.

Human hepatocytes were more sensitive than rat hepatocytes (marked difference for cytotoxicity and steatosis, slight for phospholipidosis).

Russell 1965: In vitro, AMP partially inhibited the oxidation of choline by rat liver mitochondria and completely inhibited the associated phosphorylations. Neither the oxidations nor the phosphorylations associated with β-hydroxybutyrate and succinate were affected by AMP.

Wells 1961: AMP inhibits conversion of L-methionine into lipid-bound choline in rat liver.

Åkesson 1977: In rat hepatocytes, AMP induced following effects on incorporation of phospholipid precursors into phospholipids:
- inhibition of choline incorporation (moderate compared to some structurally similar amino-alcohols);

- no effect on ethanolamine incorporation.

In additional experiments, two structurally similar amino-alcohols induced:
- effects suggesting that the inhibition of choline incorporation was of competitive nature;
- no effect on incorporation of methionine;

- change in phospholipid composition/identity: more of an unidentified phospholipid, less phosphatidylcholine.

In vivo: all below data relate to rats fed a choline-deficient (choline-free) diet. They should be interpreted with care as choline is an essential nutrient in both rats and humans.

Russell 1965: AMP was able to prevent the uncoupling (= incapable of esterifying P1) of liver mitochondria trggered by choline deficiency.

Wells 1961:

In liver, AMP inhibits synthesis of lipid-bound choline from its precursors (L-methionine, formate, glycine or DL-Serine).

Dietary choline supplementation, at a choline:AMP molar ratio of 0.2, completely prevented this inhibition.

AMP’s mechanism is not a simple competition between AMP and choline. AMP only became an inhibitor of liver choline synthesis after several days of exposure.

Longmore 1962:

1% w/w AMP into diet inhibited the conversion of phospholipid precursors into liver phospholipids, with the following inhibition magnitude and duration: ethanolamine > dimethylethanolamine and betaine > L-methionine > choline.

The following liver phospholipids were identified to be decreased by AMP: lecithin (phospholipid derived from choline) for all 5 precursors and phosphatidylethanolamine for ethanolamine (from which this phospholipid is derived).

The inhibition was not prevented by adding methyl donors (L-methionine or betaine) into the diet. The effect of dietary choline supplementation was not tested.

Overall discussion:

Choline and phosphatidyl choline are key in the transport of triglycerides from the hepatocytes in the form of VLDL (very low density lipoprotein). Interfering with the manufacture of phosphatidyl choline thus limits the capacity of the liver to transport lipids. The effect of AMP on the liver appears to be a result of interference with phospholipid synthesis in the hepatocytes. Various publications on AMP from the 1950's and 1960's have identified that it is capable of becoming incorporated into phospholipids in place of ethanolamine and/or choline and that it inhibits the uptake of choline by the liver cells. AMP also appears to inhibit the formation of choline in the liver via the conversion of ethanolamine to choline. It is possible that a phosphatidyl AMP moiety is competing for the enzyme phosphatidyl ethanolamine methyl transferase (converts phosphatidyl ethanolamine to phosphatidyl choline), preventing the formation of phosphatidyl choline, however it is as yet unclear as to the exact mechanism. What is however clear is that in the presence of sufficient dietary choline, the effects of AMP on the liver are prevented, and this is likely due to the lower dependance on de novo choline synthesis when sufficient choline is present in the diet. Thus the hepatotoxicity is in part dependent on the presence of sufficient choline in the diet.

The transport of lipids from hepatocytes is fairly consistent throughout mammalian physiology, i.e. the use of a phospholipid based transport such as VLDL. Therefore the effects observed in rats, rabbits and dogs are likely relevant to man. However the increase in hepatic lipids is only the first step in the toxicity to the liver, and further indicators of toxicity are only evident after some accumulation of lipids. There is no evidence of liver toxicity (e.g. increased liver weight, increase in liver enzymes in the blood etc.) at doses where lipid accumulation in hepatocytes is not apparent. It is also apparent that the liver toxicity is the most sensitive endpoint, occurring at doses lower than those causing other effects, such as increased post implantation loss (refer to Reproductive toxicity section).

AMP behaves as a competitive inhibitor or alternative agonist to one or more enzymes that bind and convert choline into PC that is important for VLDL production, leading to dysfunction of lipid transport, initial histological evidence of intracellular fat accumulation in vacuoles, and ultimately fatty liver accompanied by hepatocyte toxicity and pathologic changes. However, AMP-induced liver toxicity can only occur when high amounts of AMP are available to the liver relative to choline. This mode of action is consistent with animal studies using excess choline to reverse AMP effects as well as observations in humans of increased fatty liver effects among susceptible individuals who do not consume sufficient choline in their diet.

Additional information

Influence of pH adjustment on relevance of toxicology studies for hazard classification (see pH and pKa, IUCLID § 4.20/4.21):

Bibliography on maximum tolerated pH, together with pH/concentration curves for solutions of industrial and high-purity AMP grades (Fernandes, 2023), allow to conclude that repeated administration of non-neutralized AMP solutions above:

0.0005% w/w for oral route (reaching pH 9, maximum tolerated pH by oral route according to Turner, 2011), or

0.8% w/w for topical application (reaching pH 11.5, maximum tolerated pH for skin according to Shu-Hua 2020, REACH regulation 2006, ECHA 2017),

would lead to animal death or severe dose-limiting suffering. In almost all AMP in vivo toxicology studies (see IUCLID § 7.5.1, 7.5.3, 7.8), neutralization of AMP (pH adjustment or testing as AMP-HCl) allowed to test much higher concentrations and (in some studies) observe toxic effects. Since OECD guidelines do NOT require any neutralization or pH adjustment, such studies represent artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

This is confirmed experimentally by the 4 in vivo repeat-dose studies (see IUCLID §7.5.1 and 7.5.2) where AMP was tested without pH adjustment:

A 13-week oral rat study (Pittz 1977/79) was done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralization to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralizing AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

In a 5-day study on rats inhaling non-neutralized AMP aerosols (Sullivan 2017), local corrosion to skin and respiratory tract occurred at lower airborne concentrations (LOAEC: 700 mg/m3) than liver toxicity (NOEC: 700 mg/m3, LOEC: 1400 mg/m3, minimal grade liver vacuolation). pH of the 10% aerosol was calculated as 12.3 using the pH curves. Thus, protecting against local effects upon inhalation will protect against systemic effects.

In 5-day oral studies in rats and monkeys (Pittz 1977) where non-neutralized AMP reached pH >11, mortality occurred in both species. The maximum tolerated dose (MTD) in mg AMP/kg bw/day were: well below 500 in female monkeys; 500 in female rats; 1000 in male rats. No NOAEL could be set in either species due to a 10-day recovery before necropsy and liver examinations.

GHS/CLP classification is about hazard and not about risk. It aims at identifying high-dose effects of the pure substance tested in animals according to OECD guidelines (AMP being highly alkaline, this limits dose-levels and the pattern is dominated by local toxic effects). Identifying potential risks at low-dose as can exist in actual exposure conditions (where pH is balanced), is not the aim of GHS/CLP classification, but the aim of chemical risk assessment. Therefore, toxicity observed only thanks to adjustment of pH shall be ignored for classification but shall be considered for risk assessment (DNEL derivation). pH would have been >= 10.6 at the LOAEL in all animal repeat-dose toxicity studies if AMP hadn't been neutralized (see Table 1). This is largely above the maximum of pH 9 for oral route, and allows to conclude that none of the AMP adverse effects noted in animal repeat-dose studies are relevant for classification of AMP.

Ingestion (only route with adverse liver toxicity) is a negligible exposure route for REACH identified uses:

Adverse effects on liver were only observed after direct ingestion of AMP, and not in dermal or inhalative studies (see §7.5.2, 7.5.3). The liver is the first organ exposed to AMP after gastro-intestinal absorption (first-pass effect). In humans, direct ingestion only happens via the OTC drug pamabrom and such US pharma uses are out of scope of REACH/GHS/CLP. AMP is not an approved food additive in EU and is not used to treat drinking water so REACH identified uses do not involve direct ingestion of AMP. AMP is extremely water soluble, readily biodegradable and non-bioaccumulative so no significant oral exposure to AMP via environmental emissions is expected from REACH identified uses. For all these reasons, toxicology data acquired by oral route are an extreme, barely realistic worst-case of actual systemic toxicity potential by exposure routes related to REACH uses. Indeed, by skin contact and inhalation, systemic exposure potential to AMP is very limited due to:

risk management measures protecting against local corrosive effects of AMP and its industrial formulations (see IUCLID § 4.20/4.21, pH/pKa and also CSR);

negligible volatility (see IUCLID § 4.6 Vapour pressure: 45 Pa at 20°C);

few identified uses with a potential to generate aerosols (see IUCLID § 3.5 Use information);

low concentration in end products (use as pH modifier, see CSR);

only 7-17% dermal absorption through human skin (see IUCLID § 7.1.2 Dermal absorption);

there is no liver first-pass effect by these routes, as opposed to oral route.

Justification for classification or non-classification

The liver cirrhosis noted at 81 mg AMP/kg bw/day (2500 ppm AMP, tested as its neutralized form AMP-HCl) in the dog 14-week study (Lankas 1981) does not warrant STOT RE 2 classification for AMP, from regulatory and scientific points of view:

1) Effects don't meet REACH/CLP regulatory criteria:

Effects occured at 81 mg AMP/kg bw/day after 14-week exposure, and the CLP classification guidance value for STOTE RE 2 is <100 mg/kg bw/day after 13-week exposure only;

Effects occured with AMP-HCl (CAS N° 3207-12-3 list) and not AMP (CAS 124-68-5), so use for classification of AMP needs to consider the influence of the hydrochloride salt which neutralizes AMP (alkaline test item);

2500 ppm cannot be reached without AMP neutralization due to intolerable pH (calculated as 11.0-11.2 using an experimental pH curve, Fernandes 2023);

As test item neutralization is not required in OECD guidelines referenced by REACH regulation, all studies on AMP-HCl represent artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity;

This is confirmed by 13-week oral rat studies done with and without AMP neutralization (Pittz 1977/79 x2), where neutralisation artificially increased AMP’s maximum tolerated dose by a factor of at least 3.5;

The CLP classification guidance refers to rat studies, and nowhere does the CLP Regulation mention dog studies;

REACH does not request or allow testing of chemicals in dogs. Therefore, classifying chemicals based on dog studies would be unfair, discriminatory and introduce a competitive bias as no dog studies can ever be proposed or requested on any competitor substances. This would go against REACH core goals and values which include fairness, non-discrimination, and avoiding competitive bias.

2) There is no relevant hazard to human:

Effects were of limited extension and severity (very slight and scattered hepatocyte necrosis in 3/4 dogs/sex, moderate periportal fibrosis in 1 or 2/4 dogs/sex);

No indications of irreversible liver toxicity were reported in 8 studies in rats, the species cited in CLP classification guidance;

No indications of irreversible liver toxicity were reported in 3 other species (mouse, rabbit and monkey);

No indications of liver toxicity were reported in 6 human clinical trials on pamabrom (containing 25.6% w/w AMP), most of which covered sensitive populations (ill or pregnant women);

No liver effects/warnings are mentionned for any of the 20 OTC drugs containing pamabrom;

Pamabrom drugs are used since 7 decades, establishing strong pharmacovigilance.

3) There is no relevant risk to human:

A clear NOAEL for liver effects could be identified in dogs, rats and mice (insufficient investigations in rabbit and monkeys), and this, although most studies were excessively conservative as they involved neutralisation of AMP as explained above;

Ingestion is the only route with observed adverse liver toxicity and is a negligible exposure route for REACH identified uses, be it direct or indirect (via the environment);

The potential for liver exposure is negligible under realistic exposure conditions (skin contact and inhalation) based on local corrosion requiring strong risk management measures, negligible volatility, few uses generating aerosols, low concentration in end products, only 7-17% dermal absorption through human skin, and absence of liver first-pass effect;

The mechanism of AMP's liver toxicity has been thoroughly investigated and expresses only when AMP is in large excess of choline. Dietary choline supplementation can totally remove AMP’s inhibition of liver choline synthesis (Wells 1961). Therefore, AMP’s toxicity is largely indirect and has an identified antidote: choline.

Choline is an essential nutrient so it will counter-act the toxicity of any trace oral exposure to AMP. Indeed, regular and sufficient choline supply is needed for proper liver function and to avoid nonalcoholic fatty liver disease (NAFLD), a disease which is similar to AMP’s toxic effect (NIH 2022. Choline - Fact Sheet for Consumers. https://ods.od.nih.gov/factsheets/Choline-Consumer/). Recommended dietary supplies notably include: 400-425 mg/person for women from 14 years, 450 mg for pregnant women, 550 mg for breastfeeding women.

Although AMP cannot be used in food contact in EU, absence of risks upon low-dose AMP ingestion has been concluded by renowned non-EU regulatory bodies: US FDA approved AMP in various food contact applications (adhesives, coatings and fillers in paper/paperboard, certain resinous and polymeric coatings) and AMP is in China's GB 9685 positive list (additives for food contact materials).

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

Registration Dossier

Registration Dossier

Diss Factsheets

2-amino-2-methylpropanol

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Reference 1

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Reference 4

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Mode of Action Analysis / Human Relevance Framework

Additional information

Justification for classification or non-classification

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